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Bioactive phenolic compounds, metabolism and properties:a review on valuable chemical compounds in Scots pineand Norway spruce
Sari Metsamuuronen . Heli Siren
Received: 30 January 2019 / Accepted: 5 July 2019 / Published online: 22 July 2019
� The Author(s) 2019
Abstract Phenolics and extracted phenolic com-
pounds of Scots pine (Pinus sylvestris) and Norway
spruce (Picea abies) show antibacterial activity
against several bacteria. The majority of phenolic
compounds are stilbenes, flavonoids, proanthocyani-
dins, phenolic acids, and lignans that are biosynthe-
sized in the wood through the phenylpropanoid
pathway. In Scots pine (P. sylvestris), the most
abundant phenolic and antibacterial compounds are
pinosylvin-type stilbenes and flavonol- and dihy-
droflavonol-type flavonoids, such as kaempferol,
quercetin, and taxifolin and their derivatives. In
Norway spruce (P. abies) on the other hand, the main
stilbene is resveratrol and the major flavonoids are
quercetin and myricetin. In general, when the results
from the literature regarding the activities of flavonoid
glycosides and their aglycones against a total of
twenty-one microorganisms are summarized, it was
found that phenolic glycosides are less active than the
corresponding aglycones, although a number of
exceptions are also known. The aglycones in plants
respond to various kinds of biotic stress. Synergistic
effects between aglycones and their glycosides have
been observed. Minimum inhibition concentrations of
below 10 mg L-1 against bacteria have been reported
for gallic acid, apigenin, and several methylated and
acylated flavonols present in these industrially impor-
tant trees. In general, the phenolic compounds are
more active against Gram-positive bacteria, but api-
genin is reported to exhibit strong activity against
Gram-negative bacteria. The present review lists some
of the biosynthesis pathways for the antibacterial
phenolic metabolites found in Scots pine (P. sylves-
tris) and Norway spruce (P. abies). The antimicrobial
activity of the compounds is collected and compared
to gather information about the most effective sec-
ondary metabolites.
Keywords Antibacterial compounds � Metabolic
pathway � Plant enzymes �Norway spruce � Scots pine �Wood � Phenols
Abbreviations
ASE Accelerated solvent extractor
ANS Anthocyanidin synthase
CHS Chalcone synthase
CoA Coenzyme A
DFR Dihydroflavonol 4-reductase
DNA Deoxyribonucleic acid
dPCD Developmentally programmed cell death
dw Dry weight
FSI Flavone synthase
S. Metsamuuronen
Department of Chemical Technology, Lappeenranta
University of Technology, P.O. Box 20,
53851 Lappeenranta, Finland
H. Siren (&)
Department of Chemistry, University of Helsinki,
P.O. Box 55, 00014 Helsinki, Finland
e-mail: [email protected]
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Phytochem Rev (2019) 18:623–664
https://doi.org/10.1007/s11101-019-09630-2(0123456789().,-volV)( 0123456789().,-volV)
FLS Flavonol synthase
LAR Leucoanthocyanidin reductase
LDL Low density lipoproteins
MBC Minimum or minimal bactericidal
concentration
MRSA Methicillin resistant Staphylococcus aureus
MIC Minimum or minimal inhibitory
concentration
PMT 1,2 pinosylvin O-methyltransferase genes 1
and 2
PKS Polyketide synthase enzymes
SSF Solid state fermentation
STS Stilbene synthase
VRE Vancomycin-resistant enterococci
w/w Weight per weight
Background
Utilization of wood-based compounds, extracts, and
biomass has increased enormously (Rauha et al. 2000;
Jansson et al. 2013; Boden et al. 2014; Ganthaler et al.
2017). Structural components of wood (cellulose,
hemicelluloses, and lignin) and other organic sub-
stances which are integrated to biomass conversion
processes, are the basic materials of biorefineries.
Wood contains high molecular weight compounds, but
also a wide variety of low molecular mass compounds
known as extractives. The small compounds can be
separated from the high molecular and water-insoluble
wood constituents through using various kinds of
extraction techniques. Thus, the industrial use of wood
components has increased and even extracts are now
used as starting fluids and mixtures in health products
and industrial chemicals including botulin, furfural
derivatives, stilbens, tannins, flavonoids, tall oil, and
resin (Roitto et al. 2008; Royer et al. 2012; Long et al.
2013). Valuable bio-based compounds can be
obtained directly from different parts of standing trees
(Roitto et al. 2008; Royer et al. 2012; Long et al. 2013;
Siren et al. 2015; Metsamuuronen and Siren 2014;
Janusz et al. 2017; Boke et al. 2015), artificially
cultivated and fermentated mixtures (Martins et al.
2011), or isolated by-products of forest industries
(Mantau et al. 2010).
The compositions and concentrations of wood
extracts from different wood types vary a lot. The
age of wood, the harvesting time, the genetic origin,
the growing period and district all influence the
concentrations of the isolated bio compounds (Routa
et al. 2017). The concentrations are also affected by
the wood parts (needles, knots i.e. the branch bases
inside tree stems, roots, barks, heartwood, and
phloems) and the sampling positions (height, depth).
In addition, the most commonly used sample pretreat-
ment techniques have an enormous influence on the
content of extractives. Extraction systems (hot water
and supercritical fluid extractions, ultrasonication,
autoclave handling, microwaving), enzyme and
microbe treatments with reactions, and organic sol-
vents, acids or bases need to be standardized, since
they have effects on the yields of bioactive compounds
and their concentrations. Thus, the phenolic com-
pounds of low molecular weights may originate from
hydrolysis of the wood due to degradation of the
material and are not in fact the extractives from the
wood. Another determinant is the analysis method
used to identify and characterize the wood com-
pounds. When sample manipulation, such as deriva-
tization, is used to improve the sensitivity to reach the
methodological levels of the instruments, the origi-
nality of the sample matrix is lost.
Background for primary and secondary
metabolites
Scots pine (Pinus sylvestris) and Norway spruce
(Picea abies) have been chosen for the discussion of
the review, since they are the most common trees in
Northern Europe. In addition, bio refinery industry
with related industry focus interest to isolate new
chemicals for replacing polymers with bio-compounds
and investigate for reusing wastes. Extractives of pulp
and paper industry contain mostly wood species of
Scots pine (P. sylvestris) and Norway spruce (P. abies)
for new bio-based materials, energy, and bio fuel.
Nowadays, the place of renewable energy is increas-
ingly important. Biomass is the primary source of
renewable energy, and therefore the ‘‘valuable com-
pounds’’ are isolated to use only the rest material in
energy production (Gerardin 2016).
A number of chemical compounds can be isolated
from or produced in Scots pine (P. sylvestris) and
Norway spruce (P. abies). When native in the wood,
they can be classified as primary and secondary
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624 Phytochem Rev (2019) 18:623–664
metabolites. Primary metabolites have essential meta-
bolic roles in the plant. Primary metabolites are
usually described as substances that are essential
chemical units of living plant cells. These fundamental
substances are cellulose, hemicelluloses, polysaccha-
ride, and lignin. Plants synthesize a vast number of
small molecules that are secondary metabolites. In
contrast to primary metabolites, they are not necessary
for tissue growth. Secondary metabolites are formed
by evolution to defend plants against harmful attacks
by herbivores, pathogens, insects, plant-eating ani-
mals, and UV radiation (Wink 2003). Therefore, their
composition is plant-specific and genetically con-
trolled. Secondary metabolites are classified as
aliphatic compounds (terpenes and terpenoids, resin
acids, sterols, fats, waxes, fatty acids), phenolic
compounds (flavonoids, simple phenols, tannins, stil-
benes), and other secondary metabolites (sugars,
alkaloids). Phenolic compounds comprise a struc-
turally and functionally diverse group of aromatic
hydrocarbon rings, and usually at least one hydroxyl
group. In general, secondary metabolites from wood
species from temperate regions have fungicidal,
fungistatic, or bactericidal properties (Routa et al.
2017; Schultz and Nicola 2000; Treutter 2006; Gan-
thaler et al. 2017).
Usability of primary and secondary metabolites
There have been attempts to commercialize secondary
metabolites of Scots pine (P. sylvestris) and Norway
spruce (P. abies), and to manufacture health-promot-
ing pharmaceuticals, nutrients, and products for health
and welfare. Needless to say that using wood residues
on the ground is economic only, when valuable
chemical products are useful for large scale industry
production, like pine needle oil used in cosmetics and
personal care products, acyclic alcohols and essential
oils in perfumes and wood flavor compounds in food.
Bark and knotwood are the most economically
available wood residues, as they are collected in stems
and transported to saw mills and pulp mills. Then, bark
is removed from the stem and knotwood is separated
from wood chips. Unfortunately, the majority of
leaves, branches, bark, roots, and stump materials
containing valuable metabolites is wasted. Although
large amounts of bark are used in energy, pulp, and
paper production, lately stumps have also been
collected for energy production. However, usually
wood residues are left in the forest after the harvesting
of tree trunks. There is little research in literature
describing what compounds in wood parts are bene-
ficial for production and for energy industry, and in
what concentrations they exist. Removal techniques
for advantageous chemicals from stumps and thick
roots can be used to extract commercially valuable
biochemicals (Berg 2014; Hakkila 2012), like pheno-
lics, stilbenes, flavonoids, flavanones, flavonols, fla-
van-3-ols, anthocyanidins, and proanthocyanidins.
The oily tar from the Scots pine (P. sylvestris) tree
has traditionally been prepared from terpenes and
terpenoids and for thousands of years, utilized as a
preservative for timber (Holmbom 2011). Further-
more, oleoresin, terpene, and lipid components of
Scots pine (P. sylvestris) and Norway spruce (P. abies)
have been extracted as by-products in wood pulping
processes. They have been further refined to turpentine
and tall-oil and, more recently, also used for the
production of biofuel (Holmbom 2011). Phenolic
compounds are involved in resistance mechanisms as
precursors to defense-related compounds or synthesis
of polymers. They are supposed to modulate the
activity of other phytochemicals (Schultz and Nicolas
2000; Treutter 2006; Ganthaler et al. 2017). Phenolic
metabolites are considered a very important part of
both constitutive and inducible defense mechanisms
of trees (Chong et al. 2009).
Phenolic stilbenes, flavonoids, and lignans are
potential substances for biorefineries owing to their
biological properties (Conde et al. 2013; Li et al.
2012). Flavonoids have biological, nutraceutical, and
clinical effects (Maimoona et al. 2011; Li et al. 2012).
Furthermore, lignans possess chemopreventive prop-
erties (Lampe Rıos and Recio 2005; Li et al. 2012;
Saxena 2015) that are used for health and welfare
products. Lately, the interest in the phenolic com-
pounds of these conifers has increased.
Although some biologically active plants, mainly
herbaceous species, have been used as folk medicines
for centuries, much less is known about the bioactive
phenolic compounds in coniferous wood.
Stilbenes are natural defense polyphenols that
occur in many plant species. Pinosylvin (3,5-dihy-
droxy-trans-stilbene) is a naturally occurring trans-
stilbenoid, which is mainly found in heartwood of
Pinus species and exists in high concentrations in bark.
Stilbene is suggested to represent an inexpensive
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Phytochem Rev (2019) 18:623–664 625
polyphenol with considerable potential for diverse
health-promoting applications (Reinisalo et al. 2015),
like effect compounds against aging-related diseases.
Lignan has various kinds of phenylpropanoid
(propylbenzene) type molecules which are found in
all plants. Lignan is produced by secondary metabolic
pathways. Lignans such as 7-hydroxy-matairesinol,
secoisolariciresinol, lariciresinol, and nortrachelo-
genin are predominant in a large proportion of the
industrially important softwood species (Holmbom
et al. 2003). The 9-epimers of 7-hydroxy divanillyl
butyrolactol lignans, (7S,8R,89R,99R)-4,49,7-trihy-
droxy-3,39-dimethoxy-lignano-99,99-lactol and (7S,
8R,89R,99S)-4,49,7-trihydroxy-3,39-dimethoxylig-
nano-99,99-lactol have been identified and character-
ized in knotwood of Colorado spruce (Picea pungens)
(Willfor et al. 2005). Equal lignans have also been
known to occur in several spruce, pine, and fir species,
including Scots pine (P. sylvestris) and Norway spruce
(P. abies).
Metabolites in parts of trees
There is a great variability in the yields of secondary
phenolic metabolites between tree species, between
parts of the trees, and even between individuals of
different ages and botanical region (provenance)
(Kahkonen et al. 1999; Harju et al. 2003; Willfor
et al. 2003a, b, c; Venalainen et al. 2004; Hovelstad
et al. 2006; Valimaa et al. 2007; Karppanen et al. 2008;
Siren et al. 2014). The total phenolic concentrations of
76.0, 17.5, and 1.1 mg/g in gallic acid equivalents for
dried bark, needles and heartwood, respectively
(Kahkonen et al. 1999), and 6.7–13.6 mg/g in tannic
acid equivalents for wood (Venalainen et al. 2004) of
Scots pine (P. sylvestris) have been reported. Phenolic
concentrations of 10–15% and even 30%, based on dry
weight (dw) in knotwood (branch stubs embedded in
tree stems) of Norway spruce, and at least 10% dw in
knotwood of Scots pine (P. sylvestris) have been
reported (Willfor et al. 2003b, c). Several times lower
concentrations are observed in the stem wood (Willfor
et al. 2003c). A large variety of low-molecular
phenolic compounds, phenylpropanoids (tannins, lig-
nans, flavonoids, and stilbenes) are found in these
extracts, especially in the bark and knots. However,
few phenolic compounds dominate in the knotwood
extracts of most softwood species. For example, more
than half of the hydrophilic extractives of knotwood of
Norway spruce (P. abies) are lignans (Willfor et al.
2003b).
Knotwood compounds of Scots pine (P. sylvestris)
have been identified from extracts prepared into
hydrophilic organic solvents and ethanol. Earlier only
medium-large compounds with molar masses of
500–600 Da were identified due to the limitations of
analytical instruments and methods. Lately however,
high molecular mass fractions from 500 to 2200 Da
isolated from knotwood of Scots pine (P. sylvestris)
have been obtained. These compounds were mainly
oligomers of hydroxylated resin acids, especially
dehydroabietic acid, but also fatty acids, stilbenes,
and sterols. The discovery of the resin acids dimers in
native wood was a new finding, since they were not
previously identified in ethanol extracts (Smeds et al.
2018). The amounts of many lignans in softwood are
extremely low. Their amounts are less than 0.1 mg/g
(Fang et al. 2013). The fact is the sample preparation
which probably influences their identification in non-
concentrated extracts.
Metabolites in wood species
Phenolic extractives
Phenolic extracts of Scots pine (P. sylvestris) and
Norway spruce (P. abies) have been reported to
exhibit antioxidative (Kahkonen et al. 1999; Willfor
et al. 2003a; Pietarinen et al. 2006), antifungal (Harju
et al. 2003; Venalainen et al. 2004), and antibacterial
activity (Valimaa et al. 2007; Lindberg et al. 2004;
Rauha et al. 2000; Vainio-Kaila et al. 2017). A phloem
extract (Rauha et al. 2000) and a knotwood extract
from Scots pine (P. sylvestris) (Berg 2014; Lindberg
et al. 2004) as well as a needle extract from Pinus
massoniana (Feng et al. 2010) have been shown to
inhibit growth of several microorganisms, including
bacteria and yeast. Therefore, they have been sup-
posed to contain various kinds of antimicrobial
phenolic compounds, including antimicrobial pheno-
lics. Mycorrhizal fungi have long been recognized as
important microorganisms that promote tree growth
and survival (Strzelczyk and Li 2000). In Scots pine
(P. sylvestris) the major genera of endophytic bacteria
found were Methylobacterium, Pseudomonas (Pirttila
et al. 2000; Strzelczyk and Li 2000), Bacillus and
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626 Phytochem Rev (2019) 18:623–664
Paenibacillus and bacteria closely related to Bacillus
subtilis, Bacillus licheniformis, Paenibacillus spp.,
and Acinetobacter calcoaceticus (Izumi et al. 2008).
In bud of intact Scots pine (P. sylvestris), the
endophytes were identified as genera Methylobac-
terium and Pseudomonas, and the yeast as Rhodotor-
ula minuta (Pirttila et al. 2008). The trees contain
various kinds of of phenolic compounds, which have
antibacterial properties. In this case the wood struc-
tural components and extractives have been investi-
gated against methicillin-resistant Staphylococcus
aureus (MRSA) and Escherichia coli O157:H7 by
placing bacterial inoculum on surfaces for incubation
(Vainio-Kaila et al. 2017). Only the extract of Scots
pine (P. sylvestris) heartwood showed antibacterial
properties against E. coli O157:H7. The MRSA
bacteria was susceptible to extracts of Scots pine (P.
sylvestris) and Norway spruce (P. abies) species such
as heartwood, sapwood, and milled wood lignin.
General biosynthetic of phenylpropanoids
The majority of phenolic compounds in plants are
synthesized via the phenylpropanoid pathway (Iriti
and Faoro 2009). They belong to a group of pheny-
lalanine derivatives, which have a basic C6-C3 carbon
skeleton. Many of them belong to phytoalexins, which
are antimicrobial agents naturally synthesized in the
wood to respond to microorganism attacks (Ganthaler
et al. 2017). The synthesis of phytoalexins is initiated
through signal-transduction pathways linked to the
perception of pathogen receptors encoded by host
resistance genes (Dixon 2001). Phenylalanine and
tyrosine that are the precursors in phenylpropanoids
biosynthesis, are synthesized via the shikimate
(shikimic acid) pathway (Iriti and Faoro 2009), which
is present in fungi and bacteria. The general phenyl-
propanoid metabolism generates an enormous array of
secondary metabolites based on the few intermediates
of the shikimate pathway (Vogt 2010). Furthermore,
hydroxycinnamic acid and its esters are amplified in
several ways by a combination of reductases, oxyge-
nases, and transferases resulting in an organ and
developmentally specific pattern of metabolites,
which is characteristic for each plant species (Vogt
2010).
The general phenylpropanoid pathway implies that
the phenylalanine ammonia-lyase (PAL, EC 4.3.1.5)
enzyme catalyses the synthesis process by deamina-
tion of phenylalanine to cinnamic acid, which is
further catalysed by tyrosine ammonia lyase (EC
4.3.1.23) to produce p-coumaric acid. Both cinnamic
acid and p-coumaric acid are catalysed by 4-coumar-
oyl: coentzyme A (CoA) ligase (EC 6.2.1.12) to
cinnamoyl-CoA and p-coumaroyl-CoA. These phenol
products are precursors of the polyphenolic com-
pounds, which are stilbenes (C6-C2-C6 carbon skele-
ton), flavonoids (C6-C3-C6 carbon skeleton), and
lignans ((C6-C3)2 carbon skeleton) (Dixon 2001; Vogt
2010; Kodan et al. 2002; Stevanovic et al. 2009; Lim
and Koffas 2010; Tanase et al. 2018). Especially, p-
coumaroyl-CoA is a key intermediate in the biosyn-
thesis of a number of phenylpropanoids. The diversity
of phenylpropanoids is caused by hydroxylation,
methylation, acylation, isomerisation, oligomerization
and/or glycosylation (conjugation with various kinds
of carbohydrates) which modify plants secondary
metabolites to products with increased stability and
water solubility. The procedure also inactivates and
detoxificates the compounds (Gachon et al. 2005). In
general, phenylpropanoids are stored as glycosylated
forms in the vacuole, from where they can be released
and further cleaved by b-glycosidases (EC 3.2.1.x)
into their active aglycone forms e.g. against budworms
and insects (Gachon et al. 2005; Mageroy et al.
2015, 2017).
Stilbenes
Scots pine (P. sylvestris) and Norway spruce (P. abies)
synthetize different kinds of secondary metabolites.
The most important of them in P. sylvestris include
stilbenes and terpenes that defend the tree against pests
and pathogens. Their role is also to prevent rotting.
Many plant families are known to produce stilbenes,
such as Pinaceae, Gnetaceae, Myrtaceae, Poaceae,
Cyperaceae, Liliaceae, Myrtaceae, Fabaceae, Mo-
raceae, Fagaceae, Palmaceae, Polygonaceae, and
Vitaceae (see Almagro et al. 2013). No stilbenes could
be detected in Norway spruce (Hovelstad et al. 2006).
The amount of stilbenes in Scots pine varies a lot from
tree to tree (Paasela 2017).
‘‘Many plant families are known to produce stilbe-
nes, such as Pinaceae, Gnetaceae, Myrtaceae, Poa-
ceae, Cyperaceae, Liliaceae, Myrtaceae, Fabaceae,
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Phytochem Rev (2019) 18:623–664 627
Moraceae, Fagaceae, Palmaceae, Polygonaceae, and
Vitaceae (see Almagro et al. 2013)’’
Lipophilic extracts have been noticed to contain the
same major components that are found in the main
wood material. The inner bark layer contains over
10% of stilbene glucosides. Piceatannol (astringenin)
is then the main stilbene. Tannins of the proantho-
cyanidin type were extracted with non-boiling hot
water, but extraction with pressurized hot water at
140 �C or 160 �C gave non-cellulosic polysaccharides
(yields of 11–14%). These polysaccharides were
original bark basis containing pectin polysaccharides,
which are built up of arabinose, galacturonic acid, and
rhamnose. The pectin polysaccharides are of potential
interest, although they need to be studied further to
obtain more scientifically important information. The
amounts and the true chemical character of lignin are
not yet either fully elucidated (Krogell et al. 2012).
However, it is known that the inner bark contains two
times more cellulose than the outer bark, but the
opposite situation was observed for lignin, which was
determined by Klason ‘‘lignin’’ methodology (Krogell
et al. 2012). Among the potentially valuable compo-
nents, stilbene glucosides could be extracted with pure
water even at low temperatures, but tannins that are
more hydrophobic needed hot water in extraction. It
was also shown that fungal infection induced the
Norway spruce (P. abies) to produce higher levels of
stilbene synthase (STS) transcript and tetrahydroxy-
lated stilbene glycosides, and that these compounds
had antifungal activity (Hammerbacher et al. 2011).
Biosynthesis of stilbenes and stilbenoids
The biosynthesis of stilbene in Scots pine (P.
sylvestris) and Norway spruce (P. abies) (Fig. 1) is
suggested to occur in situ in the transition zone
between the sapwood and heartwood. Comparably, the
resin acids are primarily composed only in the
sapwood. The most important stilbene derivatives
are hydroxylated compounds having two phenol
moieties linked by a C2 bridge (Chong et al. 2009).
They are pinosylvin and its monomethylether, which
have important functions as phytoalexins in active
defense (Paasela 2017).
Stilbene biosynthesis starts from cinnamoyl-CoA,
p-coumaroyl-CoA and caffeoyl-CoA and ends up to
piceacides or in the presence of dihydrocinnamoyl-
CoA to dihydropinosylvin monomethyl ether.
Pinosylvin is synthetized from malonyl-CoA and
cinnamoyl-CoA with release of coenzyme A and
carbon dioxide. Biosyntheses employing cinnamic
acid as the initial point are rare compared to the more
common use of p-coumaric acid which is the most
abundant isomer of hydroxylcinnamic acid. It is
reported that pinosylvin can be isolated from heart-
wood and roots of Scots pine (P. sylvestris) (Chiron
et al. 2000).
It has been shown that a bifunctional nuclease
correlated enzyme involved in developmentally pro-
grammed cell death (dPCD) can be used as the marker
for heartwood to investigate concentrations of stilbene
and to identify biosynthesis products when investi-
gating the effect of season processes. Thus, it has been
clarified that softwood is initiated by intrinsic internal
plant based factors but not environmental factors.
Furthermore, the expression of pinosylvin-O-methyl-
transferase gene (PMT1) is not induced under stilbene-
forming conditions. Most probably, therefore PMT1 is
not involved in the stilbene pathway. However,
instead of PMT1, a new PMT-encoding gene PMT2
has been identified, that has shown an expression
pattern that is very similar to that processed with the
stilbene synthase gene. In contrast to the multifunc-
tional PMT1, PMT2 is related to methylated pinosyl-
vin with high specificity, since it follows closely the
stilbene biosynthesis (Paasela et al. 2017).
Formation of stilbenes is controlled by stilbene
synthases (EC 2.3.1.95) which are members of the
chalcone synthase superfamily of type III polyketide
synthases (Chong et al. 2009). They are classified into
p-coumaroyl-CoA- and cinnamoyl-CoA-specific
types, such as resveratrol synthase (EC 2.3.1.95) and
pinosylvin synthase (EC 2.3.1.146), which produce
resveratrol and pinosylvin, respectively (Kodan et al.
2002; Chong et al. 2009). In the biosynthesis of
stilbenes, three malonic acids are cyclized and added
to cinnamoyl-CoA or p-coumaroyl-CoA by aldol-type
cFig. 1 Biosyntheses of stilbenes commonly found in Scots pine
and Norway spruce. CoA coenzyme A; PMT pinosylvin-3-O-
methyltransferase; PSS pinosylvin synthase; STS stilbene
synthase. Collected from papers of Kodan et al. (2002), Lim
and Koffas (2010), Chong et al. (2009), and Fliegmann et al.
(1992)
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628 Phytochem Rev (2019) 18:623–664
CoAS
O
R3
R4
R2
R1
R2
R1
R4
R3
CoAS
O
Glu-O
OH
O
OROH
OH
O-Glu
OH
OO
OH
O-Glu
OROH
OH
Glu-O
Dihydrocinnamoyl-CoA
Cinnamoyl-CoA; R3=R4=Hy-Coumaroyl-CoA; R3=H, R4=OHp y
Caffeoyl-CoA; R3=R4=OH
+ 3 Malonyl-CoASTS
PSS
Pinosylvin; R1=R2=OH, R3=R4=HyResveratrol; R1-R2, R4=OH, R3=HPiceatannol; R1-R4=OH
Dihydropinosylvin;y pR1=R2=OH
Dihydropinosylviny p ymonomethyl ether;yR1=OH; R2=OMe
PMT
A
B
A
B
Methylation Glucosylation
Oligomerisation
Piceid; R1=O-Glu, R2=R4=OH, R3=H
Astringin; R1=O-Glu, R2-R4=OH
Isorhapontin; R1=R4=OH, R3=OMe, R2=O-Glu
Pinosylvin monomethyl ether;y yR1=OH, R2=OMe, R3=R4=H
Pinosylvin dimethyl ether;y yR1=R2=OMe, R3=R4=H
Isorhapontigenin;p gR1=R2=R4=OH, R3=OMe
Chemical structures of piceasidesR = H or Me
Glucosylation
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Phytochem Rev (2019) 18:623–664 629
cyclization (C2 ? C7) (Fig. 1) (Lim and Koffas
2010; Yu and Jez 2009; Flores-Sanchez and Verpoorte
2009).
In the Pinus genus, cinnamoyl-CoA is the major
substrate when pinosylvins are the most abundant
stilbenes (Gehlert et al. 1990; Zinser et al. 1998).
Besides pinosylvin (3,5-dihydroxystilbene), the syn-
thase enzyme is also able to catalyze the formation of
dihydropinosylvin from dihydrocinnamoyl-CoA. It is
not clear whether the same enzyme performs in both of
these reactions or whether two different enzymes are
involved (Fliegmann et al. 1992). In the Picea genus,
p-coumaroyl-CoA is the major substrate, since the first
step in stilbene biosynthesis is the formation of
resveratrol. Further, it can be modified by hydroxyla-
tion, methoxylation and glucosylation to produce
astringin (3,30,40,5-tetrahydroxystilbene 3-O-b-D-glu-
coside) and isorhapontin (3,40,5-trihydroxy-30-methoxystilbene 3-O-b-D-glucoside) (Hammerbacher
et al. 2011; Grunwald 2017).
Methylation of hydroxyl groups in phenyl com-
pounds can lead to increased biological activity of
stilbenes (Pezet et al. 2004). It is also observed that
under stress conditions the pinosylvin-3-O-methyl-
transferase enzyme catalyses the conversion of
pinosylvin to monomethyl ether. Particularly, the
ether form is observed in Scots pine (P. sylvestris)
rather than in Norway spruce (P. abies) (Chiron et al.
2000).
Pinosylvin-3-O-methyltransferase is not a very
specific enzyme towards pinosylvin, which is why
the enzyme may convert also other stilbenes, flavo-
noids, and simple phenolic compounds (Sandermann
et al. 2000). In addition, substitution reaction can
occur in both rings of the structure (Fig. 1): the A ring
usually has two hydroxyl groups in the meta position,
while the B ring is substituted by hydroxyl and methyl
groups in the ortho, meta and/or para positions
(Cassidy et al. 2000). In Scots pine, only the A ring
substitution has been observed. UV-light can also
convert the polyphenolic pinosylvin structures by
configurational isomerism. Photosynthesis has been
observed to activate the formation of the stereoisomer
of cis-stilbene. The extension reaction of trans–cis
isomerization produces phenanthrene, which is a
polycyclic aromatic hydrocarbon (PAH) (Kwas-
niewski et al. 2003).
The defensive role of stilbenes in conifers is well
known. Due to their antimicrobial properties, the
accumulation of large amounts of pinosylvin and
pinosylvin 3-O-methyl ether prevents the wood from
decay by microorganisms in the heartwood. It is
known that stilbenes pre-exist in wood naturally,
generally in glycosylated forms, and stilbenes that are
synthesized after microbial attack are in the free
aglycone form (Chong et al. 2009). Glycosylation of
stilbenes is postulated to protect plant cells from toxic
effects and to protect stilbenes from oxidation and
enzymatic degradation (Hipskind and Paiva 2000).
Stilbenes in Scots pine (Pinus sylvestris) and their
antimicrobial activity
In the sapwood, phloem, and needles of Scots pine (P.
sylvestris) stilbenes accumulate in the tree to defend
biotic stress after wounding, fungal attack, and ozone
intake, which increases the amount of oxygen in the
wood and enables the compound reactions chemically
with oxygen (Gehlert et al. 1990; Zinser et al. 1998;
Rosemann et al. 1991). Stilbene contents in air-dry
wood have been reported to be 0.2–2% in heartwood
and 2–8% in knots of Scots pine (P. sylvestris)
(Willfor et al. 2003c; Venalainen et al. 2004; Hovel-
stad et al. 2006). The highest concentrations have been
observed in old trees (87 years old) and at the lower
part of the stems (Hovelstad et al. 2006). Pinosylvin
and pinosylvin monomethyl ether are the most abun-
dant stilbenes in Scots pine; the other derivatives
pinosylvin dimethyl ether and dihydropinosylvin and
its methylated analogue are detected in smaller
abundance (Venalainen et al. 2004; Hovelstad et al.
2006; Valimaa et al. 2007; Pietarinen et al. 2006;
Lindberg et al. 2004).
Hydrophilic extracts of Scots pine (P. sylvestris)
have been observed to contain 22% pinosylvin and
16% pinosylvin monomethyl ether (Pietarinen et al.
2006). Pinosylvin and monomethyl pinosylvin have
been noticed to have anti-inflammatory properties,
since they reduce inflammatory gene expression and
inflammatory responses in vivo (Laavola et al. 2015).
Pinosylvin is suggested to be synthetized in plants
from cinnamic acid by ozone exposure under UV
radiation or fungal attack (Chiron et al. 2000, 2001).
The pinosylvin products have been identified in
knotwood extracts and are assumed to be significant
compounds for induced resistance in Scots pine (P.
sylvestris) (Gehlert et al. 1990; Laavola et al. 2015).
123
630 Phytochem Rev (2019) 18:623–664
Stilbenes can undergo photocyclization and photoiso-
merization processes. Especially, (Z)-stilbene may
undergo electrocyclic reactions, which help its conju-
gation and chemical affinity to toxic ligands. The
precise mechanism of the antibacterial action of
stilbenes is unclear. One possibility is that they
destroy the membrane structure, resulting in a bursting
of the cell (Nitta et al. 2002). It is suggested that
especially the two hydroxyl groups in the meta
position of the aromatic ring, and the double bond in
the carbon chain between the rings play important
roles (Valimaa et al. 2007).
Strong inhibitory effects against the growth of the
Gram-positive human pathogens Bacillus cereus, S.
aureus, and Listeria monocytogenes have been
reported (Table 1). Clear inhibition of the Gram-
negative bacteria E. coli, Salmonella infantis, and
Pseudomonas fluorescens was observed (Table 2,
Valimaa et al. 2007). The major antibacterial com-
pounds of the knotwood extract of pine, have been
found to act against the paper mill bacteria Burkholde-
ria multivorans, Alcaligenes xylosoxydans, and Bacil-
lus coagulans (Lindberg et al. 2004).
Stilbenes have been extensively studied for their
fungi-conifer interactions. A common response to
wounds or fungal attack is a decrease in glycosylation
(Viiri et al. 2001; Mala et al. 2011; Lieutier et al. 2003;
Cvikrova et al. 2008). The concentrations of stilbene
glucosides in the inner bark of Scots pine (P.
sylvestris) have been observed to decrease more in
the vicinity of fungal inoculation than in the vicinity of
mechanical damage (Viiri et al. 2001; Jyske et al.
2014). On the other hand, the concentrations of the
corresponding stilbene aglycones have been found to
be elevated only near the fungal inoculation sites (Viiri
et al. 2001).
Stilbenes in Norway spruce (P. abies) and their
antibacterial activity
Spruce bark has been found to contain high concen-
trations of several stilbene glucosides and their
aglycons, lignans, flavonoids and tannins. In Norway
spruce (P. abies) the bioactive hydroxylated stilbene
derivatives (stilbenoids) are located within phloem
and bark (Jyske et al. 2014). The current understand-
ing on the biosynthesis, distribution, and localization
of stilbenes is still fragmentary. However, the
metabolites have similar multiple biological activities
as those in pine, such as protection against environ-
mental stresses, as well as antifungal and antimicrobial
functions (Harju et al. 2003; Willfor et al. 2003a, b, c).
Three stilbene glucosides; piceid, isorhapontin, and
astringin, and the corresponding aglycones; resvera-
trol (trans-3,5,40-trihydroxystilbene, Fig. 1),
isorhapontigenin (an analogue of resveratrol), and
piceatannol (a metabolite of resveratrol) have been
found in different wood parts. Astringin and
isorhapontin are the most abundant in sapwood and
bark (Viiri et al. 2001; Zeneli et al. 2006; Danielsson
et al. 2011; Mala et al. 2011). In phenolic extracts their
proportions vary considerably, in studies of the inner
and outer phloem and bark. It was assumed that the
reason was their metabolic activity. Normalized
concentrations of 72%, 20%, and 5% were detected
for isorhapontin, astringin, and piceid from sapwood
(Zeneli et al. 2006). On the contrary, when the bark
was dry, astringin and isorhapontin concentrations
were 0.5% - 6% of its dry weight (dw) (Viiri et al.
2001; Solhaug 1990). Contrary to that, the similar
extracts from the bark were reported to contain even
47%, 39% 8%, and 0.4% isorhapontin, astringin,
piceid, and piceatannol, respectively (Zeneli et al.
2006). Analogically, stilbene glucosides have mainly
been localised in the inner bark (phloem) of the wood
(Solhaug 1990), which has been found to contain
0.7–0.8% of the above-mentioned glycosides and
* 0.05% resveratrol in fresh material (wet weight)
(Viiri et al. 2001). However, the amount of stilbene
glucosides in the mixture is high: when extracted from
bark of Norway spruce (P. abies), one cubic meter
(* 450 kg as the air-dry weight) of the spruce timber
may provide 1.6 kg of various kinds of stilbenes that is
* 0.4% of the air-dry weight (Jyske et al. 2014).
Naturally, the purification of the individual com-
pounds requires specific and sensitive methods.
Isorhapontigenin, that is an isomer of rhapontigenin
and an analogue of resveratrol (Danielsson et al. 2011)
and the stilbene glucoside dimers of piceid (Li et al.
2008) have been found to exist in extracts of Norway
spruce (P. abies) bark. The early discoveries showed
that isorhapontin (the glucoside of isorhapontigenin)
could be detected in roots (Munzenberger et al. 1990)
and root bark (Pan and Lundgren 1995). Piceaside and
piceatannol could be isolated from the other stilbenes
detected in roots (Munzenberger et al. 1990) while
piceid and astringin have been shown to exist in root
123
Phytochem Rev (2019) 18:623–664 631
Table 1 Selection of the most active antibacterial compounds and extracts from Scots pine and/or Norway spruce (MIC values of
B 300 mg/L)
Compounds in extracts and isolated
solutions
Antibacterial activity
Gram-positive bacteria Gram-negative bacteria
Stilbenes
Pinosylvin B. cereus 101 ± 6% inhibition/
106 mg L-1 (Valimaa et al. 2007)
Pinosylvin monomethyl ether S. aureus 105 ± 12% inhibition/
113 mg L-1 (Valimaa et al. 2007)
L. monocytogenes 100 ± 7% inhibition/
113 mg L-1 (Valimaa et al. 2007)
Resveratrol B. cereus MIC 50 mg L-1, E. faecalis,
MIC 50–200 mg L-1 and S. aureus MIC
100–200 mg L-1 (Paulo et al. 2010)
H. pylori MIC varying from 25 to 100 mg L-1(Paulo
et al. 2010)
Flavanones
Pinocembrin S. mutans and S. sobrinus MIC 64 mg L-1
(Danielsson et al. 2011)
S. mutans and S. orbinus MIC 64 mg L-1 (Danielsson
et al. 2011)
Dihydroflavonols
Taxifolin-7-O-rhamnopyranoside MRSA MIC 32–64 mg L-1 and MBC
64–128 mg L-1 (Bastianetto et al. 2015)
Aromadendrin-7-O-rhamnopyranoside MRSA MIC 64–128 mg L-1 and MBC
128–512 mg L-1 (Bastianetto et al.
2015)
Pinobanksin3-acetate S. mutans MIC 157 mg L-1 (Koo et al.
2002)
S. sobrinus MIC 79 mg L-1 (Koo et al.
2002)
Flavonols
Quercetin H. pylori MBC\ 200 mg L-1 (Martini et al. 2009)
3-O-methylquercetin S. aureus MIC 6.25 mg L-1 (van Puyvelde
et al. 1989)
B. subtilis MIC 50 mg L-1 (van Puyvelde
et al. 1989)
M. smegmatis, and S. pyogenes MIC
100 mg L-1 (van Puyvelde et al. 1989)
S. typhimurium MIC 50 mg L-1 (van Puyvelde et al.
1989)
E. cloaceae, E. coli, K. pneumoniae, P. vulgaris, P.
aeruginosa and P. solanacearum, S. marcescens, S.
dynasteriae MIC 100 mg L-1 (van Puyvelde et al.
1989)
Acylated kempferol-3-O-glucosides B. cereus MIC 4–16 mg L-1 (Liu et al.
1999)
S. epidermidis MIC 2–64 mg L-1 (Liu
et al. 1999)
S. aureus MIC 16–64 mg L-1 (Liu et al.
1999)
M. luteus MIC 8–16 mg L-1 (Liu et al.
1999)
Kaempferol-3-O-(200,400-di-E-p-coumaroyl)-
rhamnoside and kaempferol-3-O-(200-Z-p-
coumaroyl, 400-E-p-coumaroyl)-rhamnoside
MRSA MIC 0.5–2 mg L-1 (Otsuka et al.
2008)
E. faecalis MIC 4 mg L-1 (Otsuka et al.
2008)
E. faecium MIC 8 mg L-1 (Otsuka et al.
2008)
Myricetin MRSA, MIC 128 mg L-1 (Xu and Lee
2001)
VRE MIC 128 mg L-1 (Xu and Lee 2001)
B. cepacia, MIC 32 mg L-1 (Xu and Lee 2001)
Flavones
123
632 Phytochem Rev (2019) 18:623–664
bark only (Pan and Lundgren 1995). Needles of
Norway spruce (P. abies) contain astringin and
isorhapontin as well as piceatannol (its glucoside is
astringin) at 0.2–2% (Solhaug 1990) and 0.4% from
dw (Turtola et al. 2006), respectively.
Resveratrol (Fig. 1) is one of the most extensively
studied natural products due to its beneficial health
properties, including anti-ageing, anticancer,
antioxidant, anti-inflammatory, antiviral, cardiopro-
tective, and neuroprotective effects (Lim and Koffas
2010; Szekeres et al. 2010; Bastianetto et al. 2015).
However, its antibacterial activity has been less
studied. Resveratrol exhibits activity against Gram-
positive human pathogenic bacteria B. cereus, S.
aureus, and Enterococcus faecalis with minimum
inhibiting concentrations (MIC) of 50–200 mg L-1
Table 1 continued
Compounds in extracts and isolated
solutions
Antibacterial activity
Gram-positive bacteria Gram-negative bacteria
Apigenin B. subtilis MIC 110 mg L-1 (Oksuz et al.
1984)
E. aerugenes and E. cloaceae MIC 4 mg L-1 (Basile
et al. 1999)
P. aeruginosa MIC 8 mg L-1 (Basile et al. 1999)
P. mirabilis MIC 16 mg L-1(Basile et al. 1999)
P. vulgaris MIC 55 mg L-1 (Oksuz et al. 1984)
P. aeruginosa and E. coli MIC 110 mg L-1 (Oksuz
et al. 1984)
K. pneumoniae, S. typhi and E. coli MIC 128 mg L-1
(Basile et al. 1999)
6-Methoxyapigenin B. subtilis MIC 125 mg L-1 (Oksuz et al.
1984)
P. vulgaris MIC 125 mg L-1 (Oksuz et al. 1984)
Simple phenols
Protocatechuic acid L. monocytogenes, S. aureus and B. cereus,
MIC 24–44 mg L-1 (Chao and Yin
2009)
B. cereus MIC 100 mg L-1, MBC
200 mg L-1 (Ciric et al. 2011)
M. flavus, S. aureus and L. monocytogenes
MIC 200 mg L-1, MBC 400 mg L-1
(Chao and Yin 2009)
S. typhimurium and E. coli, MIC 24–44 mg L-1 (Chao
and Yin 2009)
P. aeruginosa and S. typhimurium MIC 100 mg L-1,
MBC 200 mg L-1 (Ciric et al. 2011)
E. coli MIC and MBC 200 mg L-1 (Chao and Yin
2009)
P. mirabilis MIC 200 mg L-1, MBC 400 mg L-1
(Chao and Yin 2009)
Gallic acid S. aureus, MIC 3.5–12.5 mg L-1 (Al-
Zahrami 2012)
S. aureus and S. epidermidis MIC
31 mg L-1 (Silva et al. 2010)
S. haemolyticus MIC 62 mg L-1 (Silva
et al. 2010)
P. mirabilis MIC 62 mg L-1 (Silva et al. 2010)
E. coli MIC 125 mg L-1 (Silva et al. 2010)
Gallic acid methyl ester S. aureus, MIC 3.5–12.5 mg L-1 (Al-
Zahrami 2012)
Picein B. cereus and E. faesalis MIC 16 mg L-1
(Sarıkahya et al. 2011)
S. aureus MIC 64 mg L-1 (Sarıkahya et al.
2011)
S. typhimurium, E. coli and P. aeruginosa MIC
32 mg L-1 (Sarıkahya et al. 2011)
K. pneumoniae MIC 64 mg L-1 (Sarıkahya et al.
2011)
p-Coumaric acid S. aureus, B. subtilis and S. pneumonia,
MIC 20 mg L-1 (Lou et al. 2012)
S. dysenteriae and S. typhimurium, MIC
10–20 mg L-1 (Lou et al. 2012)
E. coli, MIC 80 mg L-1 (Lou et al. 2012)
Lignans
(?)-Lariciresinol 40-O-glucopyranoside S. epidermidis, MIC 25 mg L-1 (Wan et al.
2012)
S. aureus, MIC 50 mg L-1 (Wan et al.
2012)
123
Phytochem Rev (2019) 18:623–664 633
Ta
ble
2S
elec
tio
no
fco
mp
ou
nd
sd
etec
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123
634 Phytochem Rev (2019) 18:623–664
Ta
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123
Phytochem Rev (2019) 18:623–664 635
Ta
ble
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nti
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123
636 Phytochem Rev (2019) 18:623–664
Ta
ble
2co
nti
nu
ed
So
ftw
oo
dP
arts
of
the
wo
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Co
mp
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123
Phytochem Rev (2019) 18:623–664 637
Ta
ble
2co
nti
nu
ed
So
ftw
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dP
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of
the
wo
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Co
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and
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nd
gre
n(1
99
5)
123
638 Phytochem Rev (2019) 18:623–664
(Paulo et al. 2010). It is less active against Gram-
negative bacteria, E. coli, Klebsiella pneumoniae and
Salmonella typhimurium than the Gram-positive bac-
teria being inactive against Pseudomonas aeruginosa
(Table 1). In another study, resveratrol has been tested
against different strains of Helicobacter pylori result-
ing in the MIC values of 25–100 mg L-1 (Paulo et al.
2011). The experiments also showed an inhibitory
effect on H. pylori urease, which is essential for the
colonialization and establishment of infections.
Resveratrol and its glucoside conjugate piceid were
also studied against three Gram-positive bacteria (B.
cereus, L. monocytogenes, and S. aureus) and two
Gram-negative bacteria (E. coli, Salmonella anatum)
(Shan et al. 2008). In general, both the aglycone and
the glucoside appeared to show similar bioactivity
resulting in the MIC values of 313 mg L-1 for S.
aureus and S. anatum and 625 mg L-1 for L. mono-
cytogenes. Regardless, resveratrol was more efficient
against B. cereus and E. coli (MIC 313 mg L-1) than
piceid (MIC 625 mg L-1). Listeria monocytogenes, S.
aureus, E. coli, and S. anatum had the same bacterio-
static concentration of 625 mg L-1, whereas that of B.
cereus was even 2500 mg L-1 for resveratrol (Shan
et al. 2008).
Trans-isorhapontin and trans-astringin have been
identified as the major, and trans-piceid as the minor
stilbene glucosides from the rootbark of Norway
spruce (P. abies) (Jyske et al. 2014). Not only stilbene
glucosides but also the corresponding stilbene agly-
cones, such as trans-resveratrol, trans-piceatannol and
trans-isorhapontigenin, have been identified from
hydrolysed extracts of spruce bark (Mulat et al.
2014). Lately, it has been confirmed that the hydrox-
ylated stilbene glucosides trans-astringin and trans-
isorhapontin are the major compounds, and that
trihydroxystilbene trans-piceid is the minor com-
pound in the roots of Norway spruce (P. abies)
(Holmbom 2011; Venalainen et al. 2004). In addition,
the aglycones of stilbene glucosides (Holmbom 2011;
Hovelstad et al. 2006) and several dimers of stilbene
glucosides have been identified in the extracts of the
roots (Valimaa et al. 2007).
Flavonoids from Norway spruce and Scots pine
Biosynthesis of flavonoids
Biosynthetic pathways (Lim and Koffas 2010; Win-
kel-Shirley 2001) of different flavonoid subclasses in
plants are shown in Fig. 2. Chalcone synthase (CHS,
EC 2.3.1.74) is the first enzyme in the flavonoid
pathway. The CHS is an enzyme confined to higher
plants and belongs to a family of polyketide synthase
enzymes (PKS) known as type III PKS (Fig. 2). It uses
three malonyl-CoA substrates and one cinnamoyl-
CoA or p-coumaroyl-CoA substrate, generating narin-
genin chalcone or pinocembrin chalcone, respectively,
in a Claisen-type cyclisation reaction (C6 ? C1) (Yu
and Jez 2009; Flores-Sanchez and Verpoorte 2009).
Following these reactions, the chalcones are isomer-
ized by chalcone isomerases (CHI, EC 5.5.1.6) to
flavanones, from which naringenin and pinocembrin
are precursors of flavones and dihydroflavonols.
Hydroxylation of flavanones at the 3 position of the
C-ring by flavanone 3b-hydroxylase (EC 1.14.11.9,
Fig. 2) leads to the formation of dihydroflavonols (He
et al. 2008; El Sayed Bashandy 2016). Flavanones are
converted to flavones by flavone synthase (FSI, EC
1.14.11.22, Fig. 2) and dihydroflavonols to flavonols
by flavonol synthase (FLS, EC 1.14.11.23, Fig. 2)
(Cheng et al. 2014). In these syntheses, a double bond
is introduced between the carbons 2 and 3 at the A
ring. Dihydroflavonol 4-reductase (DFR, EC
1.1.1.219, Fig. 2) catalyses the reduction of the
carbonyl group at the 4 position to the hydroxyl group
to give flavan-3,4-diols (leucoanthocyanidins) that are
intermediates for the biosynthesis of flavan-3-ols,
sometimes called flavanols or catechins, and antho-
cyanins (Jagannath and Crozier 2010). Next, leucoan-
thocyanidin reductase (LAR, EC 1.17.1.3, Fig. 2) or
anthocyanidin synthase (ANS, EC 1.14.11.19, Fig. 2)
converts flavan-3,4-diols into flavan-3-ols or antho-
cyanidins, respectively. Finally, anthocyanidins are
further converted to anthocyanins (i.e. glycosylated
anthocyanidins) with UDP-D-glucose:anthocyanidin
3-O-b-D-glucosyl transferase (EC 2.4.1.115).
Methylation and acylation of flavonoids
The hydroxyl groups of flavonoids are prone to
undergo methylation or glycosylation (Fig. 1).
123
Phytochem Rev (2019) 18:623–664 639
OH OH
R
OOH
OH
R2
OOH
O
R1
OH
OH
OOH
O
R1
R2
OH
OH
OOH
O
R2R1
OH
OOH
O
OHR1
R2
OH
OH
OH
O
OH
OH
R
OH
OH
OH
O+
OH
R2
OHR1
OH
OOH
O
OH
Cinnamoyl-CoA/p-Coumaroyl-CoA
+ 3 Malonyl-CoA
Anthocyanidins
R1=R2=H PelargonidinR1=OH; R2=H CyanidinR1=R2=OH; Delphinidin
OH
OH
OH
O
R1
R2
OHOH
7
CHS
R=H Pinocembrin chalconeR=OH naringenin chalcone
CHI
Flavanone
R1=H; R2=OH NaringeninR1=R2=OH EriodictyolR1=R2=H Pinocembrin
FlavoneR1=R2=OH LuteolinR1=H; R2=OH Apigenin
FSI
DihydroflavonolR1=R2=H DihydrokaempferolR1=OH; R2=H DihydroquercetinR1=R2=OH Dihydromyricetin
F3H
FLS
FlavonolR1=R2=H KaempferolR1=R2=OH MyricetinR1=OH; R2=H Quercetin
DFR
Flavan-3,4-diol(Leucoanthocyanidin)
Flavan-3-olR=H (+)-CatechinR=OH (+)-Gallocatechin
LAR
ANS
Pinobanksin
A
B
C
2
345
3´
4´
5´
Proanthocyanidins
123
640 Phytochem Rev (2019) 18:623–664
Methylation involves the transfer of the methyl group
of S-adenosyl-L-methionine to the hydroxyl group of
the flavonoid (Ibrahim et al. 1998; Kim et al. 2010). O-
Methylation of flavonoids is catalyzed by O-methyl-
transferases (EC 2.1.1.6.x, Fig. 1). They can be used
for the production of O-methylated flavonoids that
have a particular biological activity (Kim et al. 2010).
In particular, O-methylation of hydroxyl groups in
flavonoids reduces their reactivity and increases their
antimicrobial activity (Ibrahim et al. 1998). Several O-
methylated flavonols and unconjugated flavanols
(catechins) have been found in conifers.
The final step in the flavonol biosynthesis is
acylation (Fig. 3, Kaffarnik et al. 2005), which is
catalysed by enzymes that transfer the acyl group of
hydroxycinnamic acid CoA esters to flavonol 3-O-
glucosides. Three types of flavonol 3-O-glucoside
hydroxycinnamoyl transferases (EC 2.3.1.x, Fig. 3)
have been found in Scots pine (P. sylvestris) needles.
Preferentially acylation begins at position 600 followed
by position 300 (Fig. 3). In Scots pine (P. sylvestris) and
needles of Norway spruce (P. abies) flavonol 3-O-
glucosides can be esterified with ferulic or p-coumaric
acid flavonol at positions 600 and 300 (Schnitzler et al.
1996; Kaffarnik et al. 2005). Thus, the new com-
pounds are derived from carboxylic acids, in which the
hydrogen group is replaced by a hydrocarbon group.
Antimicrobial effects of various flavonoid
subclasses
Flavonoids appear widely in the plant kingdom in
many plants and they are the largest phenolic group in
nature (Lim and Koffas 2010). Flavonoids consist of a
central three-ring structure, but the various subclasses
differ from each other due to the centrally situated
heterocyclic ring structure (C-ring), where the two
benzene rings are linked together (Fig. 2). The
majority of flavonoids are flower, fruit, or leaf
pigments of different colours. It is suggested that the
antibacterial activity of flavonoids is due to their
ability to form complexes with extracellular and
soluble proteins, to make complexes with bacterial
cell walls (Cowan 1999), and to inhibit bacterial
quorum-sensing signal receptors, enzymes, and toxins
(Cushnie and Lamb 2010).
The structural diversity of flavonoid metabolites are
easily hydroxylated and methylated in presence of
catalytic enzymes in synthases, reductases, iso-
merases, and transferases reactions. Typical flavo-
noids are hydroxylated at the 5 and 7 positions of the
A-ring, whereas hydroxyl and methoxyl group substi-
tutions occur at the 30, 40, or 50 positions of the B-ring.
Like stilbenes, individual flavonoids can occur as
aglycones and glycosides. The preferred glycosylation
site on the flavonoids is the 3 position, but the 7
position is the least favourable. Almost all, natural
flavonoids exist as their O-glycoside or C-glycoside
forms in plants. It seems as though O-glycosylation
generally reduces the bioactivity of flavonoids. Nev-
ertheless, lately it has been shown that the sugar
addition to oxygen has enhanced certain types of
biological benefits in food (Xiao 2017). Glucose is the
most common sugar residue, but flavonoids with
galactose or rhamnose residues also exist in Scots
pine. Flavonoids are classified into several subgroups,
depending on the carbonyl group on the carbon 4, the
double bond between the carbon 2 and 3, the presence
of the hydroxyl group on the carbon 3, and the location
of the B ring. Six types of flavonoids (flavones,
flavanones, dihydroflavonols, flavonols, and flavan-3-
ols, and anthocyanidins) occur in Norway spruce (P.
abies) and Scots pine (P. sylvestris) trees.
Flavanones from Norway spruce and Scots pine
Two flavanones, naringenin, and eriodictyol, have
been detected in both Scots pine (P. sylvestris) and
Norway spruce. Naringenin has hydroxyl groups
attached at the 5, 7, and 40 positions (Fig. 2).
Naringenin-7-glucoside has been observed in needles
of Norway spruce (P. abies) (Slimestad et al. 1999)
and naringenin aglycone in needles of Scots pine (P.
sylvestris) (Rauha et al. 2000). Naringenin has been
reported to show strong inhibition against the Gram-
positive bacteria Micrococcus luteus, S. aureus, and
Staphylococcus epidermidis. It also shows clear
activity against B. subtilis and E. coli, and a slight
activity against P. aeruginosa (Rauha et al. 2000).
bFig. 2 Flavonoid biosynthesis. ANS anthocyanidin synthase;
CHI chalcones isomerase; CHS chalcone synthase; DFR
dihydroflavonol 4-reductase; F30H flavonoid 30-hydroxylase;
F3H flavanone 3b-hydroxylase; FLS flavonol synthase; FSI
flavone synthase; LAR leucoanthocyanidin reductase. Modified
from the paper of Lim and Koffas (2010)
123
Phytochem Rev (2019) 18:623–664 641
First, it was published that naringenin could be
isolated from Salix caprea (goat willow). Due to the
purity of the extract the results ensured that naringenin
has activity against E. coli, E. faecalis, and S. aureus
(Malterud et al. 1985). Later the antibacterial activity
of naringenin was also tested against methicillin-
resistant S. aureus (MRSA). At that time, it was
verified that the MIC values were 200–400 mg L-1
(Tsuchiya et al. 1996).
Eriodictyol has hydroxyl groups at the 5, 7, 30, and
40 positions (Fig. 2). Usually, eriodictyol exists as 70-
O-glucoside, but its 50-glucoside has been detected in
needles of Scots pine (P. sylvestris) (Larsson et al.
1992) and the 7-O-glucoside only in needles of
Norway spruce (P. abies) (Slimestad et al. 1999).
Eriodictyol has the similar structure as naringenin,
except of one additional hydroxyl group in the B-ring.
It has been shown to be bioactive against several
bacteria and it is a bitter-masking flavanone. The MIC
values were 250 mg L-1 against E. coli and B. subtilis
and 800 mg L-1 against Salmonella enterica, Pseu-
domonas putida, Listeria innocua, Lactococcus lactis,
OO
O
ROH
OH
OH O
OH OH
OH
OH
OO
O
ROH
OH
OH O
OH O
OH
OH
O
OH
OO
O
ROH
OH
OH O
OH OH
OH
O
OH
O O
OO
O
ROH
OH
OH O
OH O
OH
O
OH
O
OH
3"2"
6"
4"
Flavonol 3-O-glucosides
R = H, kaempferolR = OH, guercetinR = OMe, Isorhamnetin
3"2"
6"
4"
3"-monocoumaroylatedflavonol-3-O-glucosides
3"HCT
6"HCT
3"2"
6"
4"3"
2"
6"
4"
6"-monocoumaroylatedflavonol-3-O-glucosides
3"HCT
3", 6"-dicoumaroylatedflavonol-3-O-glucosides
Fig. 3 Suggested acylation of flavonol-3-O-glucosides. HCT hydroxycinnamoyl-CoA flavonol 3-O-glucoside hydroxycinnamoyl-
transferase. Adapted from the paper of Kaffarnik et al. (2005)
123
642 Phytochem Rev (2019) 18:623–664
and S. aureus (Mandalari et al. 2007). Pinocembrin
(Fig. 2) is an antioxidant that has hydroxyl groups
only at the 5 and 7 positions of the A-ring, while the
B-ring is unsubstituted. Pinocembrin and pinocem-
brin-7-methyl ether (pinostrobin) have frequently
been found in Scots pine (P. sylvestris) (Plant
Metabolic Network 2012). They have been noticed
to be present at dry weights below 0.02% (Willfor
et al. 2003c) in both stem wood and knots of branches
and at 0.05% in fresh needles (Rosemann et al. 1991).
Pinocembrin extracted from knotwood of pine (Pinus
cembra) has shown to inhibit the growth of both
Gram-positive (B. cereus, S. aureus, and L. monocy-
togenes) and Gram-negative (P. fluorescens, E. coli,
and Streptococcus infantis) bacteria. The strongest
activity has been observed against B. cereus (Wink
2003). The MIC values of 250 lM (64.1 lgL-1) have
been reported for commercial pinocembrin against
Streptococcus mutans and Streptococcus sorbinus
(Koo et al. 2002).
Flavones from Norway spruce and Scots pine
Apigenin (40,5,7-trihydroxyflavone, Fig. 2) and lute-
olin (30,40,5,7-tetrahydroxyflavone, Fig. 2) are fla-
vones from needles of Scots pine (P. sylvestris)
(Oleszek et al. 2002). Apigenin-7-glucoside has been
found in the needles of both Scots pine (P. sylvestris)
(Stolter et al. 2009) and Norway spruce (P. abies)
(Slimestad et al. 1999).
Twenty years ago, Basile et al. (1999) noticed that
apigenin isolated from mosses, inhibited the growth of
several Gram-negative bacteria. However, it has not
been observed to inhibit the growth of S. aureus nor E.
faecalis which are Gram-positive bacteria. It was also
reported that the MIC values were 4–128 mg L-1
against Proteus mirabilis, P. aeruginosa, Salmonella
typhi, E. coli, Enterobacter aerogenes, Enterobacter
cloaceae, and K. pneumoniae (Table 1). The MIC
values for apigenin isolated from Centaurea species
(e.g. common knapweed, Centaurea nigra), against B.
subtilis, Klebsiella pneumonia, Proteus vulgaris, P.
aeruginosa, and E. coli were higher, since they ranged
from 55 to 219 mg L-1 (Oksuz et al. 1984). However,
6-O-methylapigenin showed activity only against B.
subtilis and P. vulgaris, whereas luteolin has been
reported to inhibit the growth of MRSA with the MIC
value of 512 mg L-1 (Xu and Lee 2001).
Flavonols in Scots pine (Pinus sylvestris)
Needles of Scots pine (P. sylvestris) trees character-
istically contain kaempferol, quercetin, and their
derivatives. Kaempferol has hydroxyl groups at the
40 and 3 positions and quercetin at the 40, 50 and 3
positions (Fig. 3). Mostly, they exist in their glycoside
forms and the glucose groups have been identified to
be bonded at the 3 position. For example, kaempferol-
3-O-glucoside (astragalin) and 3-O-rhamnoside (afze-
lin) (Stolter et al. 2009; Schnitzler et al. 1997),
quercetin-3-O-glucoside (isoquercitrin), and querce-
tin-3-O-rutinoside (rutin) have been found in pine
needles (Oleszek et al. 2002; Beninger and Abou-Zaid
1997). Quercetin-3-O-rhamnoside (quercitrin) and
quercetin-3-O-galactoside (hyperocide) have been
found in twigs including needles (Stolter et al.
2009). Although all the above-mentioned quercetins
are glycosylated at the 3 position, it has also been
reported that quercetin glucosides substituted at the 30
position were dominant compounds (Oleszek et al.
2002). It has been suggested that 30-O-glucosides of
quercetin and taxifolin (dihydroflavonol) are crucial
for protecting the plant from UV light (Oleszek et al.
2002). Hence, their amounts may depend on UV
radiation.
Isorhamnetin (30-O-methylquercetin), its 3-glu-
coside, and 6-methylkaempferol-3-O-glucoside are
commonly found as methylated compounds in Scots
pine (P. sylvestris) needles (Beninger and Abou-Zaid
1997). Acetylated flavonol 3-O-glucosides have also
been frequently found (Turtola et al. 2006; Stolter
et al. 2009; Pan and Lundgren 1995) and their
concentrations have been reported to increase under
UV radiation (Schnitzler et al. 1997). Glucosides of
kaempferol, quercetin, and isorhamnetin can be
acetylated at the 300 position with p-coumaric acid
and at the 600 position with either p-coumaric acid or
ferulic acid (Kaffarnik et al. 2005). Kaempferol-3-
(dicoumaroyl)-glucoside (Stolter et al. 2009; Schnit-
zler et al. 1997) and 30,60-di-(4-coumaryl)-isorham-
netin-3-glucoside have also been identified from
needles of Scots pine (P. sylvestris). The concentra-
tions of mono- and dicoumaroyl derivatives of
isoquercitrin were measured to be 0.17–0.27% and
0.13–0.22% (dw), respectively (Turtola et al. 2006;
Stolter et al. 2009).
Kaempferol-3-O-rhamnoside, quercetin aglycone,
and their 30-O- and 7-O-glucosides have been found in
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Phytochem Rev (2019) 18:623–664 643
the bark of Scots pine (P. sylvestris) (Pan and
Lundgren 1995). The inner bark proved to contain
quercetin, 3-O-methylquercetin, and quercetin glyco-
sides, kaempferol, and some kaempferol derivatives
(Rauha et al. 2000).
Flavonols in Norway spruce (Picea abies)
In addition to kaempferol, quercetin, and isorhamnetin
derivatives, several myricetin derivatives have been
found in needles of Norway spruce (P. abies)
(Slimestad et al. 1999) e.g. laricitrin (30-O methylmyricetin) and syringetin (30,50-O-dimethyl-
myricetin). These flavonols have also been observed to
be present in the form of 3-O-glucoside, 3-(600-acetyl)-
glucoside and 3-O-rutinoside. Furthermore, kaemp-
ferol, quercetin, myricetin, and syringetin glucosides
substituted at the 7 position, as well as kaempferol-
3,40-diglucoside, myricetin-3,40-diglucoside, and
quercetin-30-glucoside, have been isolated and identi-
fied in the spruce needles. Hence, in Norway spruce
(P. abies) the glycosylation of flavonols occurs mainly
at the 3 position as glucose and rutinose complexes are
formed. From acylated flavonols, mono- and dicou-
maroyl astragalin and dicoumaroyl isorhamnetin-3-
glucoside have been identified in the needles and
quercetin-30-glucoside and isorhamnetin 3-O-(60-O-
acetyl)-glucoside in root bark (Pan and Lundgren
1995).
Dihydroflavonols
Pinobanksin (Fig. 2) is an exception among dihy-
droflavonols due to the absence of the B-ring substi-
tution (Ondrias et al. 1997; Metacyc 2019). It inhibits
peroxidation of low density lipoproteins (LDL) and it
has electron donor properties reducing a-tocopherol
radicals, of which many have vitamin E activity.
Pinobanksin-3-acetate has been reported to be active
against S. mutans and S. sorbinus with the MIC values
of 500 lM (157 mg L-1) and 250 lM (79 mg L-1),
respectively (Koo et al. 2002).
The Fig. 2 shows the dihydroflavonols found in
needles of Scots pine, which are dihydrokaempferol
(aromadendrin), dihydroquercetin (taxifolin), and
dihydromyricetin (ampelopsin). Glucosides of all
these dihydroflavonols have also been detected in
needles of Norway spruce (P. abies) (Slimestad et al.
1999). The dihydroflavonols differ from each other in
the sense that aromadendrin has a hydroxyl group at
the 40 position, taxifolin has two hydroxyl groups at
the 30 and 40 positions, and ampelopsin has three
hydroxyl groups at the 30, 40 and 50 positions in the
flavonol structure, which has the 3-hydroxyflavone
backbone. Taxifolin was found to be a powerful
antioxidant and to have antiradical activities in several
in vitro bioassays when compared with standard
antioxidant compounds (Topal et al. 2016).
In Europe, there are two Scots pine (P. sylvestris)
chemotypes with respect to the taxifolin-30-O-gluco-
side. One of the pines lacks taxifolin glucoside
whereas the other has taxifolin in needles at concen-
trations of 3–4% of dw (Laracine-Pittet and Lebreton
1988). Taxifolin-30-O-glucoside has also been
reported to exist in pine (Oleszek et al. 2002; Larsson
et al. 1992) but at much lower concentrations
(0.004–0.16% of dw). Taxifolin-30-O-glucoside with
the corresponding aglycone have also been identified
from bark of Scots pine (P. sylvestris) (Karonen et al.
2004). Generally, the (?)-chemotype glucosides have
not only been identified as taxifolin glucosides, but
also as ampelopsin-type (dihydromyricetin, Fig. 2)
and flavanone-type eriodictyol glucosides (Larsson
et al. 1992).
Antibacterial activity of flavonols
Xu and Lee (2001) investigated the relationship
between structure and activity in 38 different flavo-
noids against the multiple drug-resistant, Gram-neg-
ative rumen bacterium, Mannheimia
succiniciproducens (MBEL55E). They noticed that
flavonols are among the most active antibacterial
flavonoids and that the active flavonoids have a keto
group at the 4 position, hydroxyl groups at the 3, 5 and
7 positions, and, at least, one hydroxyl group in the
B-ring. Furthermore, the more hydrophilic flavonols
appeared to be better inhibitors than the less hydro-
philic ones.
Taxifolin-7-O-rhamnopyranoside and aromaden-
drin-7-O-rhamnopyranoside isolated from Hypericum
japonicum have been studied and been determined as
effective against isolates of S. aureus (MRSA). Ten
clinical isolates justified the MIC values at
32–64 mg L-1 for taxifolin-7-orhamnopyranoside
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644 Phytochem Rev (2019) 18:623–664
and at 64–128 mg L-1 for aromadendrin-7-O-
rhamnopyranoside. The corresponding minimum bac-
tericidal concentration (MBC) values were
64–128 mg L-1 and 128–512 mg L-1, respectively.
The MBC values were defined as the lowest concen-
tration of the tested compound that results in the death
of more than 99.9% of the bacterial population. The
possible synergy of taxifolin-7-O-rhamnopyranoside
with four conventional antibiotics ampicillin, levo-
floxacin, ceftazidime, and azithromycin has also been
investigated. An enhanced efficacy has been found,
but only with ceftazidime and levofloxacin against
clinical isolates of methicillin-resistant MRSA (An
et al. 2011).
Quercetin is the aglycone form of a number of other
flavonoid glycosides, such as rutin and quercitrin. Of
the flavonoids present in Scots pine (P. sylvestris) and
Norway spruce, quercetin (Rauha et al. 2000; Xu and
Lee 2001; Ibewuike et al. 1997; Puupponen-Pimia
et al. 2001), kaempferol (Rauha et al. 2000; Tsuchiya
et al. 1996; Xu and Lee 2001), and myricetin (Xu and
Lee 2001; Puupponen-Pimia et al. 2001) aglycones,
which are produced from the parent compound
taxifolin, were determined to have antibacterial activ-
ity. Generally, the glycosides of the flavonols were
observed to be inactive (Xu and Lee 2001; Puuppo-
nen-Pimia et al. 2001; Silva et al. 2010), although
acetylated glycosides of flavonols are observed to be
antibacterial (Si et al. 2016). However, synergistic
antibacterial activities between flavonols and their
glycosides have been observed. The inhibitory action
of quercetin was enhanced by threefold when it was
combined with guercitrin as a (1:1) mixture against
Salmonella enteritidis and B. cereus (Arima et al.
2002). In addition, an increased inhibition against B.
cereus was noticed with the combination of quercetin-
3-O-rutinoside (rutin) and quercetin as well as that
with quercetin-3-O-rutinoside and quercetin-3-O-
rhamnoside (quercitrin glycoside). Rutin and querce-
tin-3-O-galactoside (hyperoside) glycosides alone
have also indicated antibacterial activity against the
Gram-positive bacteria S. epidermidis and S. aureus
(van der Watt and Pretorius 2001).
Quercetin has exhibited antibacterial activity
against M. luteus, B. subtilis, S. aureus, S. epidermidis,
E. coli, P. aeruginosa (Rauha et al. 2000), and H.
pylori (Martini et al. 2009; Bonacorsi et al. 2012).
MIC values of 604 mg L-1 against S. aureus (Ibe-
wuike et al. 1997) and MBC of\ 200 mg L-1 against
H. pylori (Martini et al. 2009) have been reported.
3-O-Methylquercetin isolated from flowers of the
Rwandese medicinal plant (Helichrysum odoratissi-
mum) has been shown to have strong antibacterial
activity against a wide range of bacteria (van Puyvelde
et al. 1989); the MIC values from 6.25 to 100 mg L-1
were observed against several Gram-negative (E.
cloaceae, E. coli, K. pneumoniae, P. vulgaris, P.
aeruginosa, Pseudomonas solanacearum, S. typhi-
murium, Serratia marcescens, Shigella dynasteriae),
and Gram-positive (B. subtilis, Mycobacterium smeg-
matis, S. aureus, Streptococcus pyogenes) bacteria
(Table 1).
Kaempferol has shown to have a slight activity
against B. subtilis, and a clear activity against S.
aureus, but no activity against E. coli, M. luteus, or S.
epidermidis (Rauha et al. 2000). Liu et al. (1999) have
studied several acylated kaempferol-3-O-glucosides
against Gram-positive bacteria. They noticed that the
most effective compounds were those having one or
two cis-p-coumaroyl groups. Acylation was noticed to
improve the activity of kaempferol against MRSA
(Otsuka et al. 2008). The MIC values of kaempferol-3-
O-(200,400-di-E-p-coumaroyl)-rhamnoside and kaemp-
ferol 3-O-(200-Z-p-coumaroyl, 400-E-p-coumaroyl)-
rhamnoside isolated from Laurus nobilis were
0.5–2 mg L-1. The anti-MRSA activity of these
compounds was much higher than that of several
chemotherapics (oxacillin, ciprofloxacin, norfloxacin,
erythromycin, and tetracycline) and almost as high as
that of vancomycin. The MIC values of these
compounds against Enterococcus faecium and E.
faecalis were 8 mg L-1 and 4 mg L-1, respectively.
Xu and Lee (2001) have shown that flavonoids have
activity against antibiotic-resistant bacteria and
observed that myricetin was the most effective against
MRSA, vancomycin-resistant enterococci (VRE) and
Burkholderia cepacia with MIC values of
128 mg L-1, 128 mg L-1, and 32 mg L-1, respec-
tively. Furthermore, myricetin also inhibited the
growth of K. pneumoniae, whereas quercetin (MIC
256 mg L-1) and kaempferol (MIC[ 512 mg L-1)
inhibited only the growth of MRSA. It has been
determined that the myricetin activity against B.
cepacia was related to its inhibition of protein
synthesis. It has also been observed that myricetin is
the most active flavonol aglycone against different
bacteria (Puupponen-Pimia et al. 2001). In particular,
the strongest inhibition was noticed against E. coli.
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Phytochem Rev (2019) 18:623–664 645
Flavan-3-ols and anthocyanidins
The flavan-3-ols have two asymmetric carbon atoms
(chiral carbons) at the positions 2 and 3. The most
important flavan-3-ols occurring in nature are (?)-
catechin (2R,3S-3,5,7,30,40-pentahydroxyflavan) and
(?)-gallocatechin (2R,3S,-3,5,7,30,40,50-hexahydrox-
yflavan). (?)-Catechin has been found in needles
(Rosemann et al. 1991), twigs (Stolter et al. 2009),
bark (Karonen et al. 2004; Lavola et al. 2003) and root
bark (Pan and Lundgren 1995) of Scots pine (P.
sylvestris) and in needles (Jyske et al. 2014), and in
inner bark (Gehlert et al. 1990) of Norway spruce.
Catechin-3-glucoside has also been identified in bark
of Scots pine (P. sylvestris) (Pietarinen et al. 2006;
Karonen et al. 2004; Lavola et al. 2003) and 30-O-
methylcatechin has been detected in bark (Karonen
et al. 2004; Lavola et al. 2003), root bark (Pan and
Lundgren 1995) and needles (Slimestad et al. 1999) of
Norway spruce (P. abies). A strong increase in the
catechin concentration in inner bark of Norway spruce
(P. abies) has been observed, due to its response to
wounding (Lieutier et al. 2003), which suggests that
catechin has strong antimicrobial activity. Slight
activity against H. pylori has been observed for (?)-
catechin isolated from Byronima crassa (Bonacorsi
et al. 2012). The most common catechin isomer (?)-
catechin is inactive against MRSA, but generally acyl
chains at the 3 position have been observed to enhance
the anti-staphylococcal activity of the molecule (Sta-
pleton et al. 2004).
(?)-Epicatechin has been found in the bark of Scots
pine (P. sylvestris) (Karonen et al. 2004; Lavola et al.
2003) and epicatechin as well as gallocatechin in
needles of Norway spruce (P. abies) (Slimestad et al.
1999). Catechin and epicatechin have been shown to
exhibit activity against B. subtilis and E. coli (Min
et al. 2009; Idowu et al. 2010), whereas (-)-epigal-
locatechin gallate and (-)-epicatechin gallate have
shown activity against S. aureus (Akiyama et al.
2001). It has also been observed that (-)-epicatechin
gallate sensitizes MRSA to b-lactam antibiotics by
affecting the architecture and composition of the cell
wall of the MRSA bacterium (Stapleton et al. 2007).
The anthocyanin glycosides (anthocyanins)
pelargonidin-, cyanidin-, delphinidin-, and peonidin-
3- glucosides have been observed in spruce needles
(Slimestad et al. 1999). Cyanidin-3-glucoside isolated
from berry extract and extracted from pomegranate
fruit (Punica granatum) has exhibited activity against
E. coli (Puupponen-Pimia et al. 2001) and against
species of corynebacteria, staphylococci, streptococci,
B. subtilis, Shigella, Salmonella, Vibrio cholera, and
E. coli (Naz et al. 2007). In addition, pelargonidin-3-
galactoside extracted from pomegranate fruit has
exhibited similar activity (Naz et al. 2007).
Proanthocyanidins
Proanthocyanidins, also called condensed tannins, are
oligomers and polymers with the flavan-3-ol units.
They are fairly soluble in water and are able to
precipitate proteins. Proanthocyanidins can be classi-
fied into several subclasses based on the hydroxylation
and stereochemistry of the flavan-3-ol head and
extension units. The position of intermolecular link-
ages, the degree of polymerisation, branching extent,
glycosylation, and modifications such as esterification
of the 3-hydroxyl group have a specific role in the
classification (Kraus et al. 2003; Dixon et al. 2005).
Flavan-3-ol units can be linked together by A- and
B-types of linkages, in which B-type bonds are more
frequent and typically C4 ? C8, although C4 ? C6
linkages also exist (Stevanovic et al. 2009; Smolander
et al. 2012). The two main proantocyanides are
procyanidins having a dihydroxyl B-ring (3,5,7,30,40-pentahydroxylation) and prodelphinidins with a trihy-
droxyl B-ring (3,5,7,30,40,50-hexahydroxylation)
(Fig. 4). Occasionally, an additional ether bond from
O7 ? C2 or O5 ? C2 may exist, leading to double-
bonded A-type proanthocyanidins (Stevanovic et al.
2009; Hellstrom and Mattila 2008). The structures of
dimeric procyanidin and prodelphin with A-type
(elongated structure) and B type (loop structure)
linkages are presented in Fig. 4. The (epi)catechins
are the most commonly found constitutive units of the
procyanidins in temperate zone conifer heartwoods
and barks (Stevanovic et al. 2009), whereas prodel-
phinidins consists of (epi)gallocathechins. The size of
the proanthocyanidins varies from dimers to very large
polymers with average degrees of polymerization
ranging from 3 to 8 (Holmbom 2011).
Callus culture of Scots pine (P. sylvestris) has been
found to be rich in proanthocyanidins. Concentrations
of bound and free proanthocyanidins are 8.8% and
3.0% from dw, respectively (Shein et al. 2003).
Proanthocyanidins can be extracted from bark samples
123
646 Phytochem Rev (2019) 18:623–664
with hot water (Matthews et al. 1997). It was noticed
that bark of Scots pine (P. sylvestris) contains
procyanidin-type proanthocyanidins, but prodelphini-
dins were not found. It was also reported that pine bark
comprehended 3.1% of dw non-water-soluble proan-
thocyanidins and 1.0% of water-soluble procyanidins.
The latter compounds were found mainly in the inner
bark. The epicatechin/catechin-ratio of 79/23 (w/w)
and a degree of polymerisation of 5.3 were detected
for water-soluble procyanidins. Bark of Norway
spruce (P. abies) contained both types of proantho-
cyanidins and more water-soluble proanthocyanidins
than bark in Scots pine. The amounts of 0.08% and
3.6% of water-soluble prodelphinidin and procyani-
din, respectively, were detected. With an epicate-
chin/catechin-ratio of 80/20 (w/w) the degree of
polymerisation of 4.6 was detected. Proanthocyani-
dins have also been identified in needles of Scots pine
(P. sylvestris) and Norway spruce (P. abies). The
reported proanthocyanidins contents were 2.2% and
6.5% from dw, respectively (Kanerva et al. 2008).
Both prodelphinidins and procyanidin types from
monomers and dimers (molar masses of
290–610 g mol-1) to higher polymers were detected.
In contrast to the bark samples detected by Matthews
et al. (1997), needles of Scots pine (P. sylvestris) were
reported to contain more prodelphinidin than pro-
cyanidin units (Kanerva et al. 2006), which showed
the heterogeneity of the wood materials and different
recoveries in the extracts.
Tannins have been used in the tanning of leather for
their well-known ability to make complexes with
proteins. They are considered to be antibacterial
compounds as they can also denature proteins in
bacterial cell membranes. The activity of proantho-
cyanidins varies from compound to compound. Thus,
procyanidin A2 (epicatechin dimer) has been reported
to exhibit weak activity against B. subtilis (Plant
Metabolic Network 2012). However, prodelphinidin
isolated from Sericea lespedeza has been observed to
show a moderate activity against K. pneumoniae and
E. coli and a high activity against S. aureus (Min et al.
2009).
Benzoic acid and cinnamic acid derivatives
Compounds with C6-C1 and C6-C3 skeletons are
commonly classified as benzoic acids derivatives
(hydroxybenzoic acids) and cinnamic acid derivatives
(hydroxycinnamic acids). The compounds are found
in both Scots pine (P. sylvestris) and Norway spruce
(P. abies) (Fig. 5).
Biosynthesis of hydroxybenzoic acids
Several possible biosynthesis routes are proposed for
production of hydroxybenzoic (el Basyouni et al.
1964) and hydroxycinnamic acids (Reddy et al. 2005;
Koutaniemi 2007; Chen et al. 2011). The
O
O
OH
OH
R1
OH
R1OH
OH
OH
R2OH
OH
R2
O
O
O
OH
R1OH
OH
OH
R2
OH
R1OH
OHR2
R1=H ProcyanidinR1=OH Prodelphinidin
R2=OH non-esterifiedR2=galloyl esterifiedA-type B-type
A
B
2
345
6
8
42´
3´
4´
5´
C
Fig. 4 Structures of
dimeric procyanidin and
prodelphinidin with A- and
B-type linkages. Modified
from the paper of Hellstrom
and Mattila (2008)
123
Phytochem Rev (2019) 18:623–664 647
hydroxybenzoic acids include salicylic acid, benzoic
acid, p-hydroxybenzoic acid, protocatechuic acid, and
vanillic acid (Fig. 6). Especially salicylic acid (2-
hydroxybenzoic acid) can be produced by two differ-
ent biosynthesis routes (Wildermuth et al. 2001; Shah
2003). Benzoic acid-2- hydroxylase (BA2H) forms
salicylic acid from phenylalanine through cinnamic
and benzoic acids. The other route goes via a shikimic
acid pathway through chorismic and isochorismic
acids aided by isochorismate synthase (ICS) and
isochorismate pyruvate lyase (IPL).
Three alternative biosynthesis pathways have been
proposed for the formation of gallic acid (3,4,5-
trihydroxybenzoic acid) (Ishikura et al. 1984; Ossipov
et al. 2003), namely oxidation of 3,4,5-trihydroxycin-
namic acid, hydroxylation of 3,4-dihydroxybenzoic
acid (protocatechuic acid), and direct dehydrogenation
of 3-dehydroshikimic acid (Fig. 7). Most probably,
gallic acid is derived from shikimic acid (Fig. 7) by
dehydrogenation of 3-dehydroshikimic acid (Vogt
2010). This has been researched to be the most
probable pathway for gallic acid also in birch leaves
(Ossipov et al. 2003).
p-Hydroxybenzoic, salicylic, protocatechuic, vanil-
lic, gallic, and syringic acids have been detected in
mature seeds (Cvikrova et al. 2008) and in the roots of
Norway spruce (P. abies) (Mala et al. 2011; Munzen-
berger et al. 1990). Glycosides of p-hydroxybenzoic
acid and vanillic acid derivatives have been detected
in bark of Scots pine (P. sylvestris) (Karonen et al.
2004; Pan and Lundgren 1996). Especially, a callus
culture of Scots pine (P. sylvestris) has been noticed to
contain a very high concentration of p-hydroxyben-
zoic acid (31.4% of dw) (Shein et al. 2003).
Biosynthesis of hydroxycinnamic acids
Hydroxycinnamic acids are important for plant growth
and development. Therefore, they are among the most
widely distributed phenylpropanoids in plant tissues
(Rice-Evans et al. 1996). The hydroxycinnamic acids
such as p-coumaric, caffeic, ferulic, and sinapic acids
are precursors of monolignols (coniferyl alcohol,
sinapyl alcohol, and paracoumaryl alcohol) and are
directly linked to lignin biosynthesis (Fig. 7). Hydrox-
ycinnamic acid and its glycosides, as well as esters and
amides are present in various parts of forest trees and
particularly, in the cork part of the bark (Stevanovic
et al. 2009). Free and bound forms of phenolic acids,
and their esters and ethers have been detected in the
cambium and xylem of Scots pine (P. sylvestris)
(Antonova et al. 2011). In the cambium zone, the most
abundant phenolic acid was p-coumaric acid (Fig. 7)
as ether and ester forms. Sinapic acid (Fig. 8) has been
shown to dominate in the secondary thickening zone
of the xylem, both in the free form and as ester and
ether forms. It is noteworthy that caffeic and ferulic
acids (Fig. 8) have been found to be the next most
abundant phenolic acids.
In Norway spruce, ferulic and sinapic acids are
most probably synthesized from coniferylaldehyde
R1
R2R3
R4
COOH
R1
O
R2 COOH
CH3
O-Glu
O
R1HHHOHHH
Hydroxybenzoic acids
Gallic acidp-Hydroxybenzoic acidProtocatechuic acidSalicylic acidSyringic acidVanillic acid
R2OHHOHHOCH3OCH3
R3OHOHOHHOHOH
R4OHHHHOCH3H
Hydroxycinnamic acids
Caffeic acidp-Coumaric acidFerulic acidSinapic acid
R1HHHOCH3
R2OHHOCH3OCH3 Picein
Fig. 5 Structures of simple phenols found in pine and spruce
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648 Phytochem Rev (2019) 18:623–664
and sinapalaldehyde through direct oxidation by
aldehyde dehydrogenase (EC 1.2.1.68) (Koutaniemi
2007), but their formation by biosynthesis via caffeic
acid is also possible. A schematic view of both
synthesis routes from p-coumaric acid is shown in
Fig. 8. p-Coumaric acid is first catalyzed to the
corresponding CoA ester by 4-coumaric acid:CoA
ligase (EC 6.2.1.12). Then, it is converted to ester of
shikimic acid (Fig. 7) or quinic acid by hydroxycin-
namoyl-CoA:shikimic acid or hydroxycinnamoyl-
CoA:quinic acid with hydroxycinnamoyltransferase
(EC 23.1.133) and further 3- hydroxylated (EC
1.14.1336) and catalyzed (EC 23.1.133) to caffeoyl-
CoA ester (Koutaniemi 2007). The methylation of
caffeoyl-CoA to feruloyl-CoA is catalysed by caf-
feoyl-CoA O-methyltransferase (EC 21.1.104) using
S-adenosyl methionine as the methyl donor. Feruloyl-
CoA is reduced to the corresponding aldehyde by
cinnamoyl-CoA reductase (EC 1.2.1.44) (Fig. 1) and
further to coniferyl alcohol by cinnamyl alcohol
dehydrogenase (EC 1.1.1.195). Coniferaldehyde
(Fig. 8) is also a precursor for the biosynthesis of
sinapyl alcohol. It is first hydroxylated to the 5
position by ferulate-5-hydroxylase and then
methylated to the same position in catalysis by 5-
hydroxyconiferaldehyde O-methyltransferase (EC
2.1.1.68).
Antibacterial activity of small phenolic compounds
The antibacterial properties of wood are clarified by
using extractives and lignin. The phenolic compounds
from heartwood of Scots pine (P. sylvestris) have the
strongest antibacterial effect based on the extract
analyses. On the contrary, cellulose and hemicellulose
surfaces act as polysaccharide nutrition for bacteria
(Vainio-Kaila et al. 2017).
Gallic acid has shown antibacterial activity against
several bacterial species. When isolated from pome-
granate fruit (P. granatum), it has exhibited activity
against species of corynebacteria, staphylococci,
streptococci, B. subtilis, Shigella, Salmonella, V.
cholera, and E. coli (Naz et al. 2007). Gallic acid
was more active against Gram-positive than Gram-
negative bacteria. On the other hand, gallic acid was
found to be active against strains belonging to the
Gram-negative genus Shigella that consists of a group
COOH
OHCOOH
COOH
OH
COOH
OCH2
COOH
COOH
OCH2
COOH
COOH
OH
COOH
OHOH
COOH
OHOMe
Salicylic acid
Benzoic acid
BA2H
Chorismate
Shikimate pathway
Isochorismate
ICS
IPL
p-Hydroxybenzoic- acid
Protocatechuic- acid
Vanillic acid
Fig. 6 Proposed biosynthesis pathways for salicylic and
vanillic acids in plants. BA2H benzoic acid-2 hydroxylase;
ICS isochorismate synthase; IPL isochorismate pyruvate lyase.
Redrawn from papers of El Basyouni et al. (1964), Wildermuth
et al. (2001), and Shah (2003)
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Phytochem Rev (2019) 18:623–664 649
of facultative aerobic, non-spore-forming, non-motile,
and rod-shaped bacteria which are genetically closely
related to E. coli. Gallic acid has also been isolated as
one of the components from latex of Himathanthus
sucuuba (Spruce) Woodson (Apocynaceae). It has
strong antimicrobial and antibacterial activity which
has been tested against S. aureus, Staphylococcus
epidermis, Staphylococcus, haemolyticus, E. coli, and
P. mirabilis (Silva et al. 2010) with MIC values
varying from 31 to 125 mg L-1. The antibacterial
activity of gallic acid and its methyl ester showed even
stronger activity than that, since the values from 3.5 to
12.5 mg L-1 were determined (Al-Zahrami 2012). At
that time, the studies were done with different isolates
of S. aureus. Gallic acid has also been reported to
inhibit the growth of P. aeruginosa that is a common
Gram-negative, rod-shaped bacterium and causes
disease in plants, animals, and humans (Rauha et al.
2000) and H. pylori that is a Gram negative,
microaerophilic bacterium usually found in the stom-
ach, and thought to be associated to gastric ulcers
(Martini et al. 2009).
Protocatechuic acid and isovanillic acid have been
isolated from the aerial parts ofCentaurea spruneri for
antibacterial activity tests (Ciric et al. 2011). Their
MIC and MBC values against B. cereus, Micrococcus
flavus, S. aureus, L. monocytogenes, E. coli, P.
aeruginosa, P. mirabilis, and S. typhimurium were in
the range of 100–400 mg L-1. However, much
stronger bioactivity was detected, when they exam-
ined the activity of protocatechuic acid against food
spoilage bacteria S. typhimurium, E. coli, L.
O OHOH
COOH
OHOH
COOH
OH
OH
OHOH
COOH
NH2
COOH
COOH
COOH
OH
OHOH
COOH
COOH
OHOH
COOH
OHOHOH
O
O
O
OH
OH
OH
OHO
3-Dehydroshikimic acidShikimic acid
Gallic acid
Phenylalanine
Cinnamic acid
p-Coumaric acid
Protocatechuic acid
Caffeic acid 3,4,5-Trihydroxycinnamic acid
Ellagic acid
Fig. 7 Proposed biosynthetic pathways for the formation of gallic acid. Redrawn from the paper of Ishikura et al. (1984)
123
650 Phytochem Rev (2019) 18:623–664
monocytogenes, S. aureus, and B. cereus (Chao and
Yin 2009). Then the MIC ranged from 24 to
44 mg L-1.
Cinnamic, p-coumaric, caffeic, ferulic, and chloro-
genic acids (Fig. 7) isolated from berry extract have
exhibited activity against E. coli and S. enterica
(Puupponen-Pimia et al. 2001). Cinnamic acid has
also shown antibacterial activity against S. aureus and
E. aerogenes, as well as against the yeast, Candida
albicans (Nascimento et al. 2000). The bioactivity
may originate from their structure, since they all are
carboxylic acids with hydrophilic properties. It has
been determined that ferulic acid, which has a
hydroxycinnamic acid structure, has bioactivity
against B. subtilis, S. epidermis, S. aureus, and
Streptococcus pneumoniae (van der Watt and Preto-
rius 2001). As a component of lignin, ferulic acid is a
precursor of many aromatic compounds.
Chemically synthetized chlorogenic, caffeic, and
protocatechuic acids have similar inhibition against S.
mutans than those originated from wood. Their use in
medical purposes is meaningful, since S. mutans is
regarded as the main microbial agent causing dental
caries (Almeida et al. 2012). Ferulic acid and vanillic
acid (an intermediate in the production of vanillin
from ferulic acid) (Figs. 7 and 8) have been isolated
from the root bark of Onosma hispidum (Boragi-
naceae). They have been observed to be inactive
against Gram-negative bacteria and to exhibit antibac-
terial activity against E. faecalis, S. pneumoniae, S.
OH
COOH
OH
COSCoA
OHOMe
CHO
OHOMe
OH
OH
CO-shikimic acid
OH
CO-shikimic acid
OHOH
COSCoA
OHOH
COSCoA
OMe
OHOMe
CHO
OHOH
OMe
CHO
MeO
OHOH
COOH
OH
COOH
OMe
OH
COOH
OMeOHOH
COOH
OMeMeO
p-Coumaric acid
p-Coumaroyl CoA
ConiferaldehydeConiferyl alcohol
4CL
CCR
CAD
p-Coumaroylshikimic acid
C3H
Caffeoylshikimic acid
CST
Caffeoyl-CoA Feruloyl-CoA
CST CCoAOMT
5-Hydroxy-coniferaldehyde
Sinapalaldehyde
Caffeic acid Ferulic acid 5-Hydroxyferulic acid
Sinapic acid
F5H COMT
ADHG ADHG
Lignans
C3H COMT F5H COMT
Fig. 8 Proposed coniferyl alcohol and lignan biosynthesis
pathways. 4CL 4-Coumaric acid:CoA ligase; ADHG aldehyde
dehydrogenase; C3H p-coumarate 3-hydroxylase; CAD cin-
namyl alcohol dehydrogenase; CCoAOMT caffeoyl CoA O-
methyltransferase; CCR cinnamoyl CoA reductase; COMT
caffeic acid/5-hydroxyferulic acid O-methyltransferase; CST
hydroxycinnamoyl CoA:shikimic acid hydroxycinnamoyltrans-
ferase; F5H ferulate 5-hydroxylase. Redrawn from the papers of
Reddy et al. (2005), Koutaniemi (2007), and Chen et al. (2011)
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Phytochem Rev (2019) 18:623–664 651
pyogenes, and Corynebacterium diphtheria. Ferulic
acid also has bioactivity against S. aureus, S. epider-
mis, and Staphylococcus saprophyticus (Naz et al.
2007).
Commercial p-coumaric acid (Fig. 5), which is a
hydroxyl derivative of cinnamic acid (Fig. 7) has been
noticed to have comparable high activity against the
Gram-positive bacteria S. aureus, B. subtilis, and S.
pneumoniae, with an MIC of 20 mg L-1 (Lou et al.
2012). Shigella dysenteriae and S. typhimurium were
the most susceptible Gram-negative bacteria with the
MIC vales of 10 mg L-1 and 20 mg L-1, respec-
tively. In this case, E. coli was the most resistant
bacterium with the MIC of 80 mg L-1 (Table 1).
However, caffeic acid, which belongs to the group
of hydroxycinnamic acids, exhibited high antioxidant
properties, which increased when the concentration
was increased. It was also demonstrated that caffeic
acid is a stronger reducing agent in the oxidation
processes than cinnamic acid (Masek et al. 2016). It
was observed that p-coumaric acid has dual mecha-
nisms of bactericidal activity. The mechanisms disrupt
bacterial cell membranes, bind to the DNA and inhibit
related cellular functions, and ultimately lead to cell
death.
Caffeic and p-hydroxybenzoic acids were tested for
the development of antibacterial cellulose packing
material and paper hand sheets (Elegir et al. 2008). A
strong bactericidal effect was noticed against S. aureus
with a concentration of 552 mg L-1, whereas a higher
concentration was needed to kill E. coli. Phenolic
acids and flavonols as food preservatives were also
investigated (Rodrıguez Vaquero et al. 2011). The
synergistic antibacterial effect of phenolic acid mix-
tures against L. monocytogenes was observed with
combinations of gallic acid—caffeic acids, gallic
acid—protocatechuic acids, and rutin- quercetin.
The antimicrobial effects of the wood-associated
polyphenolic compounds pinosylvin, pinosylvin
monomethyl ether, astringin, piceatannol, isorhapon-
tin, and isorhapontigenin (Fig. 1) have been assessed
against both Gram-negative (Salmonella) and Gram-
positive bacteria (L. monocytogenes, S. epidermidis, S.
aureus) and yeasts (Candida tropicalis, Saccha-
romyces cerevisiae) (Plumed-Ferrer et al. 2013). In
general, the antimicrobial effects of pinosylvin were
even more prominent than those of a related stilbene
and resveratrol, which are well known for their
bioactivities. It has been stated that pinosylvin
(Fig. 1) could have potential as a natural disinfectant
or biocide in some targeted applications. (Plumed-
Ferrer et al. 2013).
Picein (Fig. 5), a glucoside of piceol (4-hydroxy
acetophenone) has been found in different parts of
Norway spruce. Non-mycorrhizal short roots of Nor-
way spruce (P. abies) have been found to contain
picein 0.09–0.2% of dry weight (Flores-Sanchez and
Verpoorte 2009). In spruce needles picein and piceol
concentrations have been 1.8–2.2% and 0.4–1.1% of
dry weight (Turtola et al. 2006; Stolter et al. 2009).
The antimicrobial potential of the isolated picein has
been examined and it has been found to exhibit
activity against both Gram-positive and Gram-nega-
tive bacteria (S. aureus, S. epidermidis, S. typhimur-
ium, E. coli, B. cereus, K. pneumoniae, E. faecalis, and
P. aeruginosa) with MIC values ranging from 16 to
64 mg L-1 (Table 1) (Sarıkahya et al. 2011).
Lignans
Softwood lignans are dimers of coniferyl alcohols
linked by b-b0-bonds. They are closely related to the
biosynthesis of lignins. In the first stage of the lignan
biosynthesis (Fig. 9), two coniferyl alcohols are stereo
selectively linked by the b-b0-bond to produce (?)-
pinoresinol (Kawai et al. 1999; Kwon et al. 2001;
Umezawa 2003). This reaction is started by a laccase
or laccase-like enzyme to produce free radicals, which
are then oriented by the protein in such a way that they
can be coupled only by the b-b0-link (Hovelstad et al.
2006).
The primary lignin, pinoresinol, undergoes a vari-
ety of conversions, such as ring cleavage, ring
formation, and substitution reactions. First, it is
enantiomer specifically transformed into lariciresinol
and secoisolariciresinol (Fig. 9), which are further
converted by dehydrogenation into matairesinol (Ste-
vanovic et al. 2009). Hydroxylation of matairesinols
leads to the formation of nortrachelogeninand hydrox-
ymatairesinol (Fig. 9), which can be transformed to a-
conidendrin (Fujita et al. 1999). The biosynthetic
pathway for many other lignans still remains unknown
(Umezawa 2003).
The knots of Norway spruce (P. abies) have been
observed to be rich in lignans (6–24% of dw), whereas
only trace amounts have been detected in the sapwood
(Willfor et al. 2003c; Hovelstad et al. 2006).
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652 Phytochem Rev (2019) 18:623–664
Hydroxymatairesinol has been reported to be the
dominant lignan (65–85% of the lignans) (Willfor
et al. 2003c; Hovelstad et al. 2006; Piispanen et al.
2008). Other lignans detected in knotwood of Norway
spruce (P. abies) are secoisolariciresinol, a-coniden-
drin, a-conidendric acid, isohydroxymatairesinol, lar-
iciresinol, lignan A, matairesinol, nortrachelogenin
(wikstromol), todolactol A, and isoliovil (Piispanen
et al. 2008). Nortrachelogenin has been found to be the
most abundant lignan in Scots pine, but small amounts
of lariciresinol, matairesinol, secoisolariciresinol, iso-
liovil, and liovil have also been determined (Willfor
et al. 2003c; Pietarinen et al. 2006). A nortrachelo-
genin content of 31% in hydrophilic extract of knot
wood has been observed (Pietarinen et al. 2006).
Lignan glucosides, xylosides, and rhamnosides have
been detected in bark of Scots pine (P. sylvestris)
(Karonen et al. 2004). Due to the many new lignan
chemicals obtained from the wood, their true struc-
tures may be incorrect until new characterization and
repeated identification is done. Earlier, it was noticed
for liovil and olivil (Willfor et al. 2005).
Although lignans have various clinically important
biological activities against bacteria (Suzuki and
Umezawa 2007), they have mostly been reported to
be inactive. Apart from nortrachhelogenin and lari-
ciresinol, lignans isolated form Scots pine (P.
sylvestris) and Norway spruce (P. abies) have been
observed to be inactive against several bacteria
(Karppanen et al. 2008). However, (?)-lariciresinol
40-O-glucopyranoside has been noticed to exhibit
strong activity against S. aureus and S. epidermidis
with the MIC values of 50 mg L-1 and 25 mg L-1,
respectively (Wan et al. 2012).
OHOH
OH
O
O
OH
OH
OMe
OMe
OH
O
OH
OH
OMe
OMe
OHOH
OH
OH
MeO
MeO
OOH
OH
MeO
MeO
O
OOH
OH
MeO
MeO
O
H
OH
OOH
OH
MeO
OMe
OHO
OOH
OH
MeO
OMe
O
Coniferyl alcohol
Pinoresinol Lariciresinol
Secoisolariciresinol
Matairesinol
2 xDP
PLRPLR
SIRD
NortrachelogeninHydroxymatairesinolConidendrin
Fig. 9 Common lignan biosynthetic pathway. DP dirigent protein; PLR pinoresinol/lariciresinol reductase; SIRD secoisolariciresinol
dehydrogenase. Redrawn from the papers of Kawai et al. (1999), Kwon et al. (2001), and Umezawa(2003)
123
Phytochem Rev (2019) 18:623–664 653
Artificially cultivated production of bioactive
compounds
The advantage in sample pre-treatment with enzyme
degradation and chemical processing is to allow
decomposition of the wood material. Then, naturally
the process prevents the real characterization of the
wood material and results in a mixture of short-chain
organic acids (Siren et al. 2015). Generally, bioactive
compounds have recovered from sources by solid–
liquid extraction using organic solvents in heat-reflux
systems (Martins et al. 2011). Solvent extraction with
acetone or hydrophilic solvents are used to isolate
lignans which are present in low concentrations in
wood. Especially, softwood knots have been
researched to contain large amounts of free aglycone
lignans (Willfor et al. 2003a, b, c, Willfor et al. 2004).
The lignan nortrachelogenin in knots of Scots pine (P.
sylvestris) (Eklund et al. 2002) and two diastereomers
of the new lariciresinol-type butyrolactone lignan,
isohydroxymatairesinol, in knots of Norway spruce
(P. abies) were identified long ago (Eklund et al.
2004). Furthermore, other techniques have also been
proposed, such as supercritical fluid extraction (SFE)
with CO2, hot water extraction (HWE), high pressure
processes, and microwave, ultrawave assisted extrac-
tion techniques, and accelerated solvent extraction
(Martins et al. 2011; Leppanen et al. 2011; Pranovich
et al. 2015).
A great deal of attention has been given to
biotechnological products, especially when they are
produced by cell cultivation technology. When opti-
mizing bioprocesses, the lack of accurate real-time
data and the different effects of physical, chemical,
and biological hydrolysis may represent a significant
bottleneck phenomenon. Interest in the development
of bioprocesses for the production or extraction of
bioactive compounds from natural sources has
increased.
Technologies of cultivation of wood materials has
been developed due to production of new enzymes that
release phenolic acids to scale-up the processes. One
of the techniques is solid state fermentation (SSF) that
is used to increase concentrations of compounds
having antioxidant activity by bioconversion of phe-
nolic compounds and mobilization of the conjugate
forms of phenolic precursors (Laavola et al. 2015;
Chao and Yin 2009; Soccol et al. 2017). SSF consists
of microbial growth and product formation on solid
particles in the absence, or near absence of water, since
the substrate itself contains sufficiently of moisture to
allow the microorganism growth and metabolism
(Jyske et al. 2014; Martins et al. 2011). Especially,
ferulic acid and vanillin could be produced by
lignolytic enzymes. The action of enzymes such as
a-amylase, laccase, and b-glucosidase, and tannin acyl
hydrolase have shown mobilization of bioactive
phenolic compounds due to SSF processes (Chao
and Yin 2009; Robledo et al. 2008). There are specific
extracellular enzymes participating in lignin degrada-
tion that are lignin peroxidase, manganese peroxidase,
and laccase (Philippoussis et al. 2009). Obviously, low
molecular weight compounds are originated from
microbial metabolism or from plant biomass and act as
diffusible oxidizing agents.
The extraction conditions can be modified con-
cerning the recovery of specific polyphenols from the
wood samples. Various sample preparation techniques
have different demands in optimization (Fang et al.
2013). Specific polyphenols can be isolated from knots
of Scots pine (P. sylvestris) by means of an accelerated
solvent extractor (ASE). In addition, hot water and
85% aqueous ethanol were good solvents for the
production of the substances. Polyphenols can be
extracted from plants by different solvent systems.
The yield depends on the extraction method, the
polarity of the solvent, and the extraction temperature
and time, just to mention a few (Xu and Chang 2007).
Aqueous ethanol mixtures are more efficient to this
purpose than the pure water and ethanol (Yilmaz and
Toledo 2006; Spigno et al. 2007). The yields are high,
but the selectivity for phenolic compounds is low.
An industrial knotwood sample from a pulp mill
was sequentially extracted with cyclohexane and
ethanol/water (95:5) in a large-scale Soxhlet equip-
ment. The total amount could be recovered by the
initial extraction with nonpolar solvents. Sequential
extraction has been the most efficient way to extract
pinosylvin, since the lipophilic compounds have
already been removed. It has been calculated that the
purity of pinosylvin was high (16%, w/w) with
sequential extraction. On the other hand, the yields
in hot-water extracts were much lower than obtained
in extraction with polar solvents. It was noticed that
methanolysis enabled isolation of high-molar-mass
compounds in the water extracts, which had been
assumed to be polymeric or oligomeric hemicellu-
loses. When alkaline carbonate or hydroxide solutions
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654 Phytochem Rev (2019) 18:623–664
are needed in extraction processes applied conditions,
it may limit the use of such solutions in industrial
applications.
Without seasoning, fresh woods from Norway
spruce (P. abies) and bark, phloem, and heartwood
and from Scots pine (P. sylvestris) have been identified
to contain lactic, citric, succinic, and adipic acids,
which are considered as source chemicals in the
biopolymer industry. The total acid concentration and
specifically the amounts of acetic and succinic acids in
fluids of bark, phloem, and heartwood of pine were
58.4 g/kg and 3.5–6.9 g/kg, respectively. In spruce,
the most dominant acids were l-lactic and l-malic
acids. Traditionally, disassembling of trees for lignin
degrading occurs with peroxidases and laccases
enzymes. However, biological pretreatment of organic
material is done with microorganisms, which inter alia
can be yeast or brown-, white-, and soft-rot fungi (Isroi
et al. 2011).
In biological processes, the starting wood material
may also be treated by hydrolyzation with a strong
mineral acid such as sulfuric acid. In that case,
cellulose degrades to hexoses. Simultaneously, the
wood hemicelluloses degrade to acetic acid and
pentose, which are further changed to furans with an
extension reaction. Thereafter, the reaction goes to
furfural and other wood-based compounds. Further-
more, wood lignin goes to phenols and thereafter to
phenolic compounds of low molecular weights (Balat
2011).
Lignocelluloses can be processed to biochemicals
and fuels with pretreatment, hydrolysis, fermentation,
and product separation (Alvira et al. 2010; Kim et al.
2004; Lee et al. 2002; Mosier et al. 2005). Currently,
lignocelluloses are converted via saccharification
followed by biochemical or catalytic process to sugars.
The process is time-consuming because it still needs
either acid or enzyme hydrolysis.
Syneregistic effects of compounds from Scots pine
(Pinus sylvestris) and Norway spruce (Picea abies)
Antibacterial effect can derive from various com-
pounds with antibacterial properties or from a syner-
gistic effect of several compounds (Metsamuuronen
and Siren 2014). The recent research (Vainio-Kaila
2017) showed that generally pine is more antibacterial
than spruce, although spruce is also found to have
antibacterial effects against most bacterial strains. The
extracts of Scots pine and Norway spruce stem, bark,
roots, leaves and needles contain both phenolic
extracts and essential oils which are bacteriostatic
against several bacteria. Extracts from several other
wood species besides Scots pine and Norway spruce
have been studied in regard to their health effects.
In the case of tuberculosis, several natural com-
pounds of low mammalian toxicity and strong in vitro
activity exist (Pauli and Schilcher 2004). In addition,
for first time promising antibacterial and antifungal
effects of epidihydropinidine (Fyhrquist et al. 2019),
which is the major piperidine alkaloid in the needles
and bark of Norway spruce (P. abies) showed
inhibitory against bacterial and fungal strains with
the lowest MIC value of 5.37 mg L-1 against P.
aeruginosa, E. faecalis, Candida glabrata, and C.
albicans. In addition, for epidihydropinidine (Fyhr-
quist et al. 2019), which is the major piperidine
alkaloid in the needles and bark of Norway spruce (P.
abies) showed mild inhibitory against bacterial and
fungal strains with the lowest MIC value of
5.37 mg L-1 against P. aeruginosa, E. faecalis, C.
glabrata, and C. albicans.
Summary
Scots pine (P. sylvestris) and Norway spruce (P. abies)
stem, bark, roots, and needles contain several phenolic
secondary metabolic compounds that exhibit antibac-
terial activity against several microbes. Most of them
are produced via the phenylpropanoid pathway by the
involvement of several plant enzymes, synthases,
transferases, reductases, and hydroxylases.
Flavonols and their derivatives are among the most
abundant flavonoids in both Scots pine (P. sylvestris)
and Norway spruce. From stilbenes, Norway spruce
(P. abies) contains resveratrol and its derivatives,
whereas Scots pine (P. sylvestris) contains different
pinosylvins. The compounds having MIC values of
10 mg L-1 or less have been recognised as potential
new medicines (Rıos and Recio 2005). Such com-
pounds found in Scots pine (P. sylvestris) and Norway
spruce (P. abies) are methylated and acylated deriva-
tives of the flavonols quercetin, kaempferol, gallic
acid, and methyl ester of gallic acid. The activity of the
compounds has in general been higher against Gram-
positive bacteria than Gram-negative bacteria. The
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Phytochem Rev (2019) 18:623–664 655
flavone, apigenin has been reported to be the most
active compound against Gram-negative bacteria with
MIC values ranging from 4 to 128 mg L-1.
Synergistic effects between individual compounds
have been observed to increase their activity, which is
comprehended through the multi-component defense
system of the plants. There are several other less
antibacterial compounds in softwoods (Scots pine, P.
sylvestris and Norway spruce, P. abies) that in
combination with others can be useful as antibacterial
agents. Apart from drugs, natural antibacterial com-
pounds may be exploited in the protection of food or
different surfaces. For example, protecatechuic acid
has been observed to effectively inhibit the growth of
food poisoning bacteria B. cereus, E. coli, S.
typhimurium, and L. monocytogenes. Most wood-
derived materials (branches, roots, bark, needles, and
stems) containing valuable metabolites are dis-
charged. However, utilization of these materials is
difficult. High yields of highly valuable compounds
are required in order for it to be economical to collect
and transport these tissues, to extract, and to purify the
compounds. The metabolite yield can also be affected
by the delay between harvesting and processing.
Acknowledgements Open access funding provided by
University of Helsinki including Helsinki University Central
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